Stephen G. Shaughnessy

Reamed versus nonreamed intramedullary nailing of lower extremity long bone fractures: a systematic overview and meta-analysis.

OBJECTIVE To determine the effect of reamed versus nonreamed intramedullary (IM) nailing of lower extremity long bone fractures on the rates of nonunion, implant failure, malunion, compartment syndrome, pulmonary embolus, and infection. DESIGN Quantitative systematic review of prospective, randomized controlled trials. DATA IDENTIFICATION MEDLINE and SCISEARCH computer searches provided lists of published randomized clinical trials from 1969 to 1998. Extensive hand searches of major orthopaedic journals, bibliographies of major orthopaedic texts, and personal files identified additional studies. STUDY SELECTION AND DATA EXTRACTION Of 676 citations initially identified, sixty proved potentially eligible, of which four published and five unpublished randomized trials met all eligibility criteria. Each of three investigators assessed study quality and abstracted relevant data. RESULTS The pooled relative risk of reamed versus nonreamed nails (nine trials, n = 646 patients) was 0.33 [95% confidence interval (CI), 0.16 to 0.68; p = 0.004]. The absolute risk difference in nonunion rates with reamed IM nailing was 7.0 percent (95% CI, 1 to 11 percent). Thus, one nonunion could be prevented for every fourteen patients treated with reamed IM nailing [number needed to treat (NNT) = 14.28]. The risk ratios for secondary outcome measures were: implant failure, 0.30 (95% CI, 0.16 to 0.58; p < 0.001); malunion, 1.06 (95% CI, 0.32 to 3.57); pulmonary embolus, 1.10 (95% CI, 0.26 to 4.76); compartment syndrome, 0.45 (95% CI, 0.13 to 1.56); and infection, 0.98 (95% CI, 0.21 to 4.76). Sensitivity analyses suggested that reported rates of nonunion and implant failure were higher in studies of lower quality. The type of long bone fractured (tibia or femur), the degree of soft tissue injury (open or closed), study quality, and whether a study was published or unpublished did not significantly alter the relative risk of nonunion between reamed and nonreamed IM nailing. CONCLUSIONS There is evidence from a pooled analysis of randomized trials that reamed IM nailing of lower extremity long bone fractures significantly reduces rates of nonunion and implant failure in comparison with nonreamed nailing.

High and low pressure pulsatile lavage of contaminated tibial fractures : an in vitro study of bacterial adherence and bone damage

OBJECTIVE This study was designed to examine the effect of pulsatile irrigation on microscopic bone architecture and its time-dependent efficacy in removing adherent slime-producing bacteria from cortical bone. DESIGN Using an in vitro model, ten-millimeter transverse cut sections from five human tibiae were contaminated with Staphylococcus aureus and subjected to either high pressure pulsatile lavage (HPPL; seventy pounds per square inch, normal saline) or low pressure pulsatile lavage (LPPL; fourteen pounds per square inch, normal saline) or served as controls. Alteration of bony architecture was quantified by using a previously described ordinal scale and histomorphometric analysis of each transverse cut section of tibia. To assess the time-dependent effectiveness of pulsatile lavage in removing adherent bacteria from bone, ten-millimeter transverse cut sections from ten canine tibiae were contaminated with S. aureus and subjected to high or low pressure pulsatile lavage immediately or after one, three, or six hours. Scanning electron microscopy and bacterial cultures were used to assess the removal of adherent bacteria. RESULTS HPPL resulted in significantly greater macroscopic damage than was seen with LPPL or in controls (ANOVA, p < 0.001). Histomorphometry revealed that HPPL was associated with significantly larger and more numerous fissures or defects in the cortical bone when compared with low pressure irrigation (p < 0.001). However, high and low pressure lavage were associated with similar degrees of periosteal separation from the cortical bone surface (p = 0.87). Both high and low pressure lavage were effective in removing adherent bacteria from bone at three hours irrigation delay, but only high pressure lavage removed adherent bacteria from bone at six hours delay. CONCLUSION In this in vitro study, compared with HPPL, LPPL led to less structural damage and was equally effective in removing bacteria within three hours debridement delay; however, the efficacy of LPPL at six hours debridement delay is questionable. This finding may have clinical significance in the development of infection following open tibial fractures.

Homocysteine-dependent Alterations in Mitochondrial Gene Expression, Function and Structure HOMOCYSTEINE AND H2O2 ACT SYNERGISTICALLY TO ENHANCE MITOCHONDRIAL DAMAGE

Mitochondrial abnormalities have been identified in hepatocytes of patients with hyperhomocysteinemia and in endothelial cells from the aortas of rats with diet-induced hyperhomocysteinemia. However, the mechanism by which homocysteine affects mitochondria is unknown. In this report, homocysteine-induced expression of the mitochondrial electron transport chain gene, cytochrome coxidase III/ATPase 6,8 (CO3/ATPase 6,8), was identified in a human megakaryocytic cell line DAMI using mRNA differential display. Steady-state mRNA levels of CO3/ATPase 6,8, as well as other mitochondrial transcripts, were increased in DAMI cells by homocysteine in a concentration- and time-dependent manner. Despite an increase in mitochondrial RNA levels and changes in mitochondrial ultrastructure, no effect on either cell growth or mitochondrial respiration rates was observed in DAMI cells exposed to homocysteine at concentrations up to 1 mm. In contrast, 1 mmhomocysteine in the presence of Cu2+, which is known to generate H2O2, significantly decreased mitochondrial RNA levels, caused gross morphological changes in mitochondrial ultrastructure, and inhibited both cell growth and mitochondrial respiration rates. However, precursors of cellular glutathione and preexposure to heat shock blocked the decrease in mitochondrial RNA levels caused by homocysteine and Cu2+. The observations that (i) homocysteine and H2O2, but not H2O2alone, caused a decrease in mitochondrial RNA levels, (ii) intracellular levels of H2O2 were significantly increased in the presence of homocysteine and Cu2+, and (iii) catalase, but not free radical scavengers, prevented a decrease in mitochondrial RNA levels, provide evidence that homocysteine and H2O2 act synergistically to cause mitochondrial damage. Furthermore, our findings suggest that intracellular glutathione and heat shock proteins play a role in protecting mitochondria against the adverse effects elicited by homocysteine and H2O2.

Cancer cell interactions with injured or activated endothelium

Blood vessels and lymphatics are the most important pathways for dissemination of cancer cells but the entry and exit of these cells into and from the vasculature requires that they pass through barriers formed by the endothelium and its basement membrane. This review summarizes evidence that this step in metastasis can be regulated by microenvironmental influences which alter the properties of this barrier. These phenomena can be attributed to both ‘passive’ and ‘active’ responses of the endothelium. The microvasculature is susceptible to perturbation from environmental agents, host cells and cancer cells. There is clinical and experimental evidence that this can upregulate the metastic process. Using established animal models of pulmonary microvascular injury it has been shown that endothelial damage promotes the localization and metastasis of circulating cancer cells to the lung and that this effect is lost after endothelial repair. Oxidative stress is an effector of vascular damage in several of the experimental models. While endothelial cells appear to be directly susceptible to free radical attack, basement membranes are not. However, oxidative injury of endothelial cells causes release of proteases which can then degrade the basement membrane. This event is associated with generation of tumor cell chemoattractants and enhances cancer cell invasion of vascular basement membranesin vitro. Vascular endothelial cells are also susceptible to stimulation by systemic mediators including cytokines, thrombin, or endotoxin which induce a series of active responses in the vessel wall. These perturbed endothelial cells synthesize and express cell surface adhesion molecules which can interact with cancer cells. They also release chemoattractants which stimulate cancer cell motility. We postulate that such responses endow the vessel wall with the potential to act as a determinant of metastatic rate.

Effect of Leptin on Vascular Calcification in Apolipoprotein E–Deficient Mice

Objective—The adipocytokine leptin has been proposed to increase cardiovascular risk in both obese and diabetic individuals. In the current study, therefore, we used apoE-deficient mice to examine the effects of leptin on both lesion size and calcification. Methods and Results—Mice were treated with once daily intraperitoneal injections of leptin (125 &mgr;g/mouse/d) for 2 months. The mice were then euthanized, and sections of the aortic root and thoracic aorta analyzed histomorphometrically. Measurements of lesion size and surface area occupied by atherosclerotic lesions did not reveal any differences between nontreated and leptin-treated animals. However, von Kossa staining of the aortic root demonstrated an 8.3±2.0-fold increase in lesion calcification as well as a 2.5±0.6-fold increase in valvular calcification in those animals treated with leptin. In addition, the percent total lesion area demonstrating ALP-positive staining was 5.4±2.1-fold greater in leptin-treated mice when compared to nontreated control mice. This increase in ALP staining was also accompanied by an increase in the expression of the osteoblast-specific markers, osteocalcin, and osteopontin. Conclusions—Based on these observations, we conclude that leptin may increase cardiovascular risk by promoting osteogenic differentiation and thus vascular calcification.

Oxidized low‐density lipoprotein promotes osteoblast differentiation in primary cultures of vascular smooth muscle cells by up‐regulating Osterix expression in an Msx2‐dependent manner

We have previously shown that oxidized low‐density lipoproteins (oxLDLs) act synergistically with β‐glycerophosphate to induce the osteogenic differentiation of primary bovine aortic smooth muscle cells (BASMCs). In the present study, we attempt to resolve the mechanism responsible for this effect by examining the expression of several osteoblast‐specific transcription factors. Thus, by culturing BASMCs in the absence or presence of β‐glycerophosphate and/or oxLDL, we demonstrate that β‐glycerophosphate induces both Runx2 and Osterix (Osx) expression. In contrast, oxLDL has no effect on Runx2 expression but rather it enhances β‐glycerophosphate‐induced osteoblast differentiation by further up‐regulating Osx expression. In an attempt to elucidate the mechanism responsible for this latter effect, we examined the ability of oxLDL to affect Msh homeobox 2 (Msx2) expression. Similar to its effect on Osx expression, oxLDL was found to synergistically enhance β‐glycerophosphate‐induced Msx2 expression in an extracellular signal‐regulated kinase 1 and 2 (Erk 1 and 2)‐dependent manner. Furthermore, oxLDLs ability to enhance both β‐glycerophosphate‐induced Osx expression and alkaline phosphatase activity was prevented when the BASMCs were first transfected with Msx2‐specific siRNA. Taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteoblast differentiation and vascular calcification in vivo. J. Cell. Biochem. 112: 581–588, 2011.

Oxidized low-density lipoprotein acts synergistically with β-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells

Previous studies have localized osteoblast specific markers to sites of calcified atherosclerotic lesions. We therefore decided to use an established in vitro model of vascular calcification in order to confirm earlier reports of oxidized low‐density lipoprotein (oxLDL) promoting the osteogenic differentiation of vascular smooth muscle cells. Treatment of primary bovine aortic smooth muscle cells (BASMCs) with β‐glycerophosphate was found to induce a time‐dependent increase in osteoblast differentiation. In contrast, no effect was seen when BASMCs were cultured in the presence of oxLDL alone. However, when the BASMCs were cultured in the presence of both β‐glycerophosphate and oxLDL, β‐glycerophosphates ability to induce osteoblast differentiation was significantly enhanced. In an attempt to resolve the mechanism by which this effect was occurring, we examined the effect of β‐glycerophosphate and oxLDL on several pathways known to be critical to the differentiation of osteoblasts. Surprisingly, β‐glycerophosphate alone was found to enhance Osterix (Osx) expression by inducing both Smad 1/5/8 activation and Runx2 expression. In contrast, oxLDL did not affect either Smad 1/5/8 activation or Runx2 activation but rather, it enhanced both β‐glycerophosphate‐induced Osx expression and osteoblast differentiation in an extracellular signal‐regulated kinase 1 and 2 (Erk 1 and 2) ‐dependent manner. When taken together, these findings suggest a plausible mechanism by which oxLDL may promote osteogenic differentiation and vascular calcification in vivo. J. Cell. Biochem. 105: 185–193, 2008.

Long-term treatment with sodium warfarin results in decreased femoral bone strength and cancellous bone volume in rats

The issue of whether long-term sodium warfarin therapy results in decreased bone density is controversial. To address this question, we randomized rats to once daily subcutaneous injections of either sodium warfarin (0.20 or 0.25 mg/kg) or saline for 28 days and monitored the effects on bone, both biomechanically and by histomorphometric analysis. In addition, the anticoagulant status of both saline- and warfarin-treated rats were monitored throughout the course of the experiment by measuring the prothrombin time, expressed as international normalized ratios (INRs). Rats treated with 0.25 mg/kg warfarin demonstrated INRs of approximately 2.6, while rats treated with either 0.20 mg/kg warfarin or saline were found to have INRs of 1.3 and 1.0, respectively. Biomechanical testing of the right femur of rats treated with 0.25 mg/kg warfarin demonstrated that warfarin caused an 8% reduction in bone strength as measured by maximum tolerated load. A similar reduction in the biomechanical parameters of energy to break (P<.0001) and force at break point (P<.005) was also observed. Histomorphometric analysis of the left femur of warfarin-treated rats revealed a 17% reduction in cancellous bone volume. This was accompanied by a 60% decrease in osteoblast surface, as well as an 80% reduction in osteoid surface. In contrast, warfarin treatment had the opposite effect on osteoclast surface, which was 35% higher following warfarin treatment. Based on these observations, we conclude that clinically relevant doses of warfarin decrease femoral bone strength and cancellous bone volume, both by decreasing the rate of bone formation and increasing the rate of bone resorption.

The binding of unfractionated heparin and low molecular weight heparin to thrombin-activated human endothelial cells.

The binding of unfractionated heparin to endothelium is thought to be responsible for the rapid and saturable phase of unfractionated heparin clearance. Thrombin can induce endothelial cells to express and/or secrete a number of heparin binding proteins that have the potential to increase the binding of unfractionated heparin and to a lesser extent the binding of low molecular weight heparin. To explore this possibility, we examined the binding of unfractionated heparin and low molecular weight heparin to thrombin-activated endothelial cells. Cultured human umbilical vein endothelial cells were used to determine the binding of 125I-labeled unfractionated heparin and low molecular weight heparin to untreated and to thrombin-activated cells. After thrombin treatment, we obtained a time-dependent increase in the binding of radio-labeled unfractionated heparin. In contrast, there was much less binding of low molecular weight heparin, and a time-dependent increase was not apparent. After 30, 45, and 60 minutes of thrombin treatment, the binding of unfractionated heparin was significantly higher than that of low molecular weight heparin. The increase in binding of unfractionated heparin to thrombin-activated cells also was demonstrated using fluorescently labeled unfractionated heparin followed by fluorescence microscopy. The average fluorescence intensity of thrombin-treated cells increased by 44% when compared with resting cells. The present results indicate that thrombin can increase the binding of unfractionated heparin to human umbilical vein endothelial cells. Thus, an activated endothelium may contribute to the variability of the anticoagulant response to unfractionated heparin. In contrast, the binding of low molecular weight heparin is much less affected, which may account for its better bioavailability and longer half-life.

Leptin promotes osteoblast differentiation and mineralization of primary cultures of vascular smooth muscle cells by inhibiting glycogen synthase kinase (GSK)-3β.

In this study, we begin to investigate the underlying mechanism of leptin-induced vascular calcification. We found that treatment of cultured bovine aortic smooth muscle cells (BASMCs) with leptin (0.5-4 μg/ml) induced osteoblast differentiation in a dose-dependent manner. Furthermore, we found that leptin significantly increased the mRNA expression of osteopontin and bone sialoprotein, while down-regulating matrix gla protein (MGP) expression in BASMCs. Key factors implicated in osteoblast differentiation, including members of the Wnt signaling pathway, were examined. Exposure to leptin enhanced phosphorylation of GSK-3β on serine-9 thereby inhibiting activity and promoting the nuclear accumulation of β-catenin. Transfection of BASMCs with an adenovirus that expressed constitutively active GSK-3β (Ad-GSK-3β S9A) resulted in a >2-fold increase in GSK-3β activity and a significant decrease in leptin-induced alkaline phosphatase (ALP) activity. In addition, qRT-PCR analysis showed that GSK-3β activation resulted in a significant decrease in the expression of osteopontin and bone sialoprotein, but a marked increase in MGP mRNA expression. When taken together, our results suggest a mechanism by which leptin promotes osteoblast differentiation and vascular calcification in vivo.