The effect of systemic administration of sclerostin antibody in a mouse model of distraction osteogenesis.

Distraction osteogenesis (DO) is a successful technique for bone lengthening, but one problem is the need to keep an external fixator in place until bone completely regenerates. We hypothesized that the systemic administration of sclerostin antibodies (Scl-Ab) can accelerate bone regeneration in a mouse model of DO. A total of 110 mice were randomized to receive one intravenous injection per week of either Scl-Ab (100 mg per kg body weight) or saline after DO surgery. Mice were sacrificed on day 11, 17, 34 or 51 post-surgery. Microcomputed tomography showed that bone volume per tissue volume of the Scl-Ab treated group was significantly higher on day 11 (P=0.009). Histological examinations indicated that chondrocytes and fibrocartilage predominated in the Scl-Ab group at day 11. The radiographic score of bone healing was also higher in Scl-Ab treated animals at day 11. There was a trend towards higher ultimate force and work to failure in Scl-Ab treated groups on day 34 and 51 (P>0.05). These data suggest the potential utility of Scl-Ab to reduce the time during DO when an external fixator is required.

Overview of the radiology, histology, and bone morphogenetic protein expression during distraction osteogenesis of the mandible.

INTRODUCTION: Distraction osteogenesis (DO) is a form of in vivo tissue engineering during which an osteotomy and controlled distraction are used to lengthen bone. The molecular signals that govern distraction-induced bone formation have not been fully elucidated. Specifically, the role of bone morphogenetic proteins (BMPs) in DO of the mandible remains unclear. OBJECTIVE: To characterize the radiologic and histologic evolution of newly formed bone during DO of the mandible and to relate these changes to the expression of BMPs. METHODS: Fourteen skeletally mature male rabbits were used. A distractor device was surgically applied to one side of the mandible following osteotomy. After 1 week (latency period), distraction was started at a rate of 0.25 mm every 12 hours for 3 weeks (distraction period) and was followed by a 3-week consolidation period. Two animals were sacrificed each week after surgery (weeks 1 to 7). The mandible was resected and the new bone assessed by radiography and histology. The expression of BMPs was also analyzed using immunohistochemistry. RESULTS: There was radiographic and histologic evidence of bone formation during the distraction period. By week 6, there was mature woven bone within the distraction zone. Bone morphogenetic proteins 2 and 4 were strongly expressed in osteoblasts during distraction and in chondrocytes during consolidation. The expression of BMP-7 was relatively minor. CONCLUSION: The temporal and spatial pattern of BMP expression suggests that these proteins are important mediators of mandibular DO. Understanding the expression of BMPs may facilitate the use of recombinant proteins to enhance the rate and quality of bone generation during craniofacial DO.

Overexpression of Bcl-2 attenuates apoptosis and protects against myocardial I/R injury in transgenic mice.

To test whether the antiapoptotic protein Bcl-2 prevents apoptosis and injury of cardiomyocytes after ischemia-reperfusion (I/R), we generated a line of transgenic mice that carried a human Bcl-2 transgene under the control of a mouse alpha-myosin heavy chain promoter. High levels of human Bcl-2 transcripts and 26-kDa Bcl-2 protein were expressed in the hearts of transgenic mice. Functional recovery of the transgenic hearts significantly improved when they were perfused as Langendorff preparations. This protection was accompanied by a threefold decrease in lactate dehydrogenase (LDH) released from the transgenic hearts. The transgenic mice were subjected to 50 min of ligation of the left descending anterior coronary artery followed by reperfusion. The infarct sizes, expressed as a percentage of the area at risk, were significantly smaller in the transgenic mice than in the nontransgenic mice (36.6 +/- 5 vs 69.9 +/- 7.3%, respectively). In hearts subjected to 30 min of coronary artery occlusion followed by 3 h of reperfusion, Bcl-2 transgenic hearts had significantly fewer terminal deoxynucleodidyl-transferase nick-end labeling-positive or in situ oligo ligation-positive myocytes and a less prominent DNA fragmentation pattern. Our results demonstrate that overexpression of Bcl-2 renders the heart more resistant to apoptosis and I/R injury.

Overexpression of CuZnSOD in coronary vascular cells attenuates myocardial ischemia/reperfusion injury.

Superoxide dismutase scavenges oxygen radicals, which have been implicated in ischemia/reperfusion (I/R) injury in the heart. Our experiments were designed to study the effect of a moderate increase of copper/zinc superoxide dismutase (CuZnSOD) on myocardial I/R injury in TgN(SOD1)3Cje transgenic mice. A species of 0.8 kb human CuZnSOD mRNA was expressed, and a 273% increase in CuZnSOD activity was detected in the hearts of transgenic mice with no changes in the activities of other antioxidant enzymes. Furthermore, immunoblot analysis revealed no changes in the levels of HSP-70 or HSP-25 levels. Immunocytochemical study indicated that there was increased labeling of CuZnSOD in the cytosolic fractions of both endothelial cells and smooth muscle cells, but not in the myocytes of the hearts from transgenic mice. When these hearts were perfused as Langendorff preparations for 45 min after 35 min of global ischemia, the functional recovery of the hearts, expressed as heart rate x LVDP, was 48 +/- 3% in the transgenic hearts as compared to 30 +/- 5% in the nontransgenic hearts (p <.05). The improved cardiac function was accompanied by a significant reduction in lactate dehydrogenase release from the transgenic hearts. Our results demonstrate that overexpression of CuZnSOD in coronary vascular cells renders the heart more resistant to I/R injury.

Mechanism of transforming growth factor-beta1-induced expression of vascular endothelial growth factor in murine osteoblastic MC3T3-E1 cells.

Transforming growth factor-beta1 (TGF-beta1), an abundant growth factor in bone matrix, has been shown to be involved in bone formation and fracture healing. The mechanism of action of the osteogenic effect of TGF-beta1 is not clearly understood. In this study, we found that the addition of TGF-beta1 to murine osteoblastic MC3T3-E1 cells induced vascular endothelial growth factor (VEGF) mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of TGF-beta1. The induction was superinduced by cycloheximide and blocked by actinomycin D. Ro 31-8220, a protein kinase C inhibitor, abrogated the induction. In addition, curcumin, an inhibitor for transcription factor AP-1, also blocked the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappaB. Transient transfection experiment showed that VEGF promoter activity increased 3.6-fold upon TGF-beta1 stimulation. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after TGF-beta1 stimulation. Our results therefore suggest that at least part of the osteogenic activity of TGF-beta1 may be attributed to the production of VEGF.

Upregulation of vascular endothelial growth factor by H2O2 in rat heart endothelial cells.

Hydrogen peroxide (H2O2) is a reactive oxygen species generated by several metabolic pathways in mammalian cells. Endothelial cells are extremely susceptible to oxidative stress. H2O2 has been reported to increase the permeability in these cells. Using rat heart endothelial cell culture as a model system, we examined the effect of H2O2 on the gene expression of vascular endothelial growth factor (VEGF), a potent mitogen of endothelial cells and a vascular permeability factor. By Northern blot analysis we found that VEGF mRNA responded to H2O2 in a dose-and time-dependent manner. The induction was superinduced by cycloheximide and blocked by actinomycin D. N-Acetylcysteine, a synthetic antioxidant, was able to suppress the induction. H7, a protein kinase C inhibitor, could also block the induction. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors, AP-1 and NF-kappaB. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 4 h after H2O2 stimulation. Our results demonstrate that VEGF gene expression is upregulated by H2O2 in these endothelial cells.

Upregulation of vascular endothelial growth factor by angiotensin II in rat heart endothelial cells.

Vascular endothelial growth factor (VEGF) is a potent mitogen for endothelial cells and a vascular permeability factor. In this study we found that the addition of angiotensin II (AII) to rat heart endothelial cells induced VEGF mRNA production. VEGF mRNA levels reached a plateau within 2 h after the addition of AII and decreased after 4 h. The induction was superinduced by cycloheximide and blocked by actinomycin D. Losartan, an AT1 receptor antagonist, abolished the induction of VEGF mRNA by AII, whereas PD 123319, an AT2 receptor antagonist, had no effect on VEGF mRNA induction. H7, a protein kinase C inhibitor, blocked the induction. RT-PCR experiments showed two mRNA species (VEGF 120 and VEGF 164) in these cells and both species were stimulated by AII. Transient transfection experiment showed that VEGF promoter activity was increased 2.2-fold upon AII stimulation. Electrophoretic mobility shift assay revealed an enhanced binding of transcription factors AP-1 and NF-kappa B. Immunoblot analysis showed that the amount of secreted VEGF was elevated in the medium 8 h after AII stimulation. Our results demonstrate for the first time that the upregulation of VEGF by AII may play a significant role in AII-induced hyperpermeability.

Complications of limb-lengthening in children who have an underlying bone disorder.

We retrospectively reviewed the results, particularly with regard to complications, of lengthening of long bones in eight children (nine limb segments) who had a limb-length discrepancy secondary to an underlying bone disorder (Group 1). The mean age of these patients was twelve years (range, six to sixteen years), the mean preoperative limb-length discrepancy was 6.0 centimeters (range, 2.7 to 8.8 centimeters), and the mean lengthening of the nine limb segments was 6.2 centimeters (range, 2.7 to 9.0 centimeters). Only two extremities were equalized. We compared the results in Group 1 with those of limb-lengthening in seven children (nine limb segments) who had a discrepancy secondary to post-traumatic growth arrest (Group 2) and seven children (seven limb segments) who had a discrepancy secondary to growth arrest following an infection in the bone (Group 3). All of the procedures were performed at our institution during the same time-period by the same surgeons. There were forty-one complications (twenty-five minor and sixteen major), with a mean of five complications per limb segment, in Group 1; twenty-six complications (twenty minor and six major), with a mean of three complications per limb segment, in Group 2; and twenty-two complications (fourteen minor and eight major), with a mean of three complications per limb segment, in Group 3. The results in Group 1 suggest that the Ilizarov technique for lengthening, although effective in restoring the length of the extremity, is associated with a higher rate of complications in patients who have a discrepancy due to an underlying bone disorder than in those who have a discrepancy due to growth arrest. Therefore, caution should be exercised before a lengthening procedure is recommended for a patient who has an underlying bone disorder.

Chondrodiatasis in a patient with spondyloepimetaphyseal dysplasia using the Ilizarov technique: successful correction of an angular deformity with ensuing ossification of a large metaphyseal lesion. A case report.

Distraction through the physis (chondrodiatasis) is a controversial technique with unpredictable results. However, it has been used in the past for the lengthening and correction of angular deformities of long bones. We report the case of an 11-year-old patient with spondyloepimetaphyseal dysplasia (SEMD) who presented with a severe recurvatum deformity of the left proximal tibia secondary to collapse of the tibial plateau into a large metaphyseal cystic lesion. Using the chondrodiatasis technique with a percutaneously applied Ilizarov circular frame, we were able to correct this deformity. Surprisingly, healing and ossification of the metaphyseal lesion was simultaneously observed at the end of the treatment, a finding which, to the best of our knowledge, has not been previously reported.

Management of fibular hemimelia: amputation or limb lengthening.

We reviewed retrospectively 22 patients (23 limb segments) with fibular hemimelia treated by amputation or limb lengthening to evaluate these methods of treatment. There were 12 boys and 10 girls, all with associated anomalies in the lower limbs. Twelve patients (13 limb segments) had early amputation and prosthetic fitting and ten had tibial lengthening using the Ilizarov technique. At the latest follow-up, the twelve patients who had amputation were functioning well and had few complications. The ten patients who had lengthening had suffered numerous complications, and all had needed either further corrective surgery or to wear braces or shoe-raises. Two of the ten lengthened limbs required late amputation for poor function or cosmesis. There were fewer hospital admissions, clinic visits, and periods of absence from school in the amputation group. Our findings suggest that amputation is a more effective method of management than limb-lengthening in severe fibular hemimelia. The Ilizarov method is an attractive alternative for selected patients, but its exact role is not yet established. One problem is that families often have unrealistic expectations of the surgical and prosthetic technology available and may refuse amputation when this has been recommended.

[Histologic evaluation of bone regeneration in cases of limb lengthening by Ilizarov's technique. An experimental study in the dog]. / Evaluation histologique de la régénération osseuse dans les cas d'allongement des membres suivant la technique d'Ilizarov. Une étude expérimentale chez le chien.

The histology of bone regeneration in cases of limb lengthening was studied in eight adult dogs. Following an osteotomy of the right fibula, an Orthofix (four dogs) or an Ilizarov external fixator (4 dogs) was installed and an osteotomy of the right tibia performed. Lengthening was started seven days after the surgery at the rate and rhythm of 0.5 mm every 12 hours for three weeks and was then followed by compression of 1 mm. The animals were then sacrificed in pairs 3, 6, 9 and 12 weeks after the start of lengthening. Histological evaluation of regenerate bone was performed using hematoxylin and eosin, trichrome and Von Kossa stain (decalcified). New bone at the site of distraction seemed to be formed mostly through intramembranous, and to a lesser extent, through endochondral ossification as evident by the presence of cartilaginous islands within the distraction gap of old specimens. However, these foci of cartilage cells did not have the appearance of growth plates. From the third week of lengthening, collagen fibers were laid down along the longitudinal axis of distraction. Mineralization of these fibers started at the bone ends and during the following weeks, progressed towards the center of the distraction gap. New bone was formed from both the medullary cavity and the periosteum.

Subacute hematogenous osteomyelitis: are biopsy and surgery always indicated?

Forty-four consecutive cases of subacute osteomyelitis admitted at our institution over a 12-year period were retrospectively reviewed to assess the effectiveness of conservative versus surgical treatment of this condition and to determine the indications for open biopsy and surgical debridement. Twenty-four cases were treated with antibiotics only, and 20 had surgical debridement followed by antibiotics. Except for one case that received inadequate antibiotic therapy, all patients responded well to this treatment, whether conservative or surgical. At an average follow-up of 18 months, there were no recurrences. Our results also showed that with a careful radiologic assessment of these cases, most lesions showed characteristic benign radiologic features. We can therefore conclude that conservative management of cases of subacute osteomyelitis is as effective as surgical treatment. We believe that conservative treatment with antibiotics should be the first line of management in most of these cases and that open biopsy or surgical debridement or both should be reserved for cases that do not respond to antibiotics or show aggressive radiologic features.

Clinical features and pharmacologic treatment of Paget's disease.

Paget's disease of the bone is characterized by a focal increase in the rate of bone turnover, which goes through phases of activity and quiescence. Most patients are asymptomatic. The two cardinal features are pain and deformities, and many complications may arise. Diphosphonates and calcitonin are the main therapeutic modalities.

Reducing polypharmacy in extended care.

In this 5-year prospective study, we determined the feasibility of reducing polypharmacy in a long-term care institution by a systematic review of the pharmacy records. At 6-month intervals, the computer printout of all medications prescribed to patients in a 550-bed institution was reviewed. After patients taking more than 10 different drugs were identified, their physician was notified and was asked to review their medications according to specific guidelines. The number of patients taking 10 or more medications was reduced from 67, when the program was started, to 9. The average number of medications per patient was reduced from 5.5 to 4.6. This program reduced the prevalence of polypharmacy and had long-lasting effects on the physicians' prescribing habits. We also believe it led to improved patient care by reducing the potential for drug interactions and to cost savings for the pharmacy.

Investigation of the influence of acetylsalicylic acid on the steady state of long-term therapy with theophylline in elderly male patients with normal renal function.

The risk inherent in the clinical control of patients with theophylline is widely recognized. Elderly patients may present an additional risk because of altered pharmacokinetics and the use of concomitant medication. Acetylsalicylic acid has been proposed for primary and secondary prevention of myocardial infarction and possible strokes. This investigation was undertaken to determine if concomitant administration of acetylsalicylic acid in elderly patients would alter steady-state levels of theophylline. A population of smoking male patients older than 60 years of age under long-term control of chronic obstructive pulmonary disease (COPD) with theophylline were evaluated for a baseline period of 3 days. Serum levels were measured at 6:00 AM and 6:00 PM. An enteric-coated acetylsalicylic acid preparation, 650 mg by mouth, was added to the daily slow-release theophylline, 6:00 AM hour dose regimen for 4 weeks. The serum levels of theophylline and salicylates were measured at 6:00 PM after dosing and at 6:00 AM the following day, at weekly intervals for 4 weeks. Urine specimens collected before administration of medication at 6:00 AM were analyzed for salicylates to further confirm dosage compliance. All volunteers continued to be clinically controlled throughout the treatment period and no symptoms of either overdose or underdose of either medication occurred. Plateau or trough theophylline serum levels did not change significantly during the salicylate treatment period. Salicylate serum levels did show during treatment self-induced metabolism. It is concluded that in elderly male patients, a daily concomitant therapeutic salicylate regimen does not alter steady-state serum theophylline levels and therefore does not per se necessitate the assay of theophylline blood levels in elderly patients.

Clinical presentation of Paget's disease of the bone in older patients.

To determine the clinical presentation and manifestations of Paget's disease of the bone in patients older than 60 years, we reviewed the cases of 56 patients attending a bone clinic. Pain was the presenting symptom in 34 cases. It was attributed to the disease process in 21 cases, to osteoarthritis in 11, to trigeminal neuralgia in 1, and to osteosarcoma in 1. Other clinical manifestations included deformities (15 cases), diminished mobility and unsteady gait (9), hearing impairment (7), lethargy (4), diminished vision (3), cognitive deficit (3), sense of warmth in limbs (2), ill-fitting dentures (1), and fracture (1). We concluded that in patients older than 60 years, Paget's disease of the bone may present itself in a variety of ways, some of which may be mistakenly attributed to the "aging process" or some other disease. Furthermore, in this age group, osteoarthritis is responsible for the pain experienced by about one third of symptomatic patients.

Aluminum toxicity and Alzheimer's disease. Is there a connection?

Similarities between Alzheimer's disease and aluminum toxicity have led to the suggestion that a causal relationship may exist. However, the two disorders differ in many respects. The author reviews the evidence concerning the relationship between aluminum toxicity and Alzheimer's disease and answers the question, is there a connection?