Direct percutaneous gene delivery to enhance healing of segmental bone defects

BACKGROUND: Healing of segmental bone defects can be induced experimentally with genetically modified osteoprogenitor cells, an ex vivo strategy that requires two operative interventions and substantial cost. Direct transfer of osteogenic genes offers an alternative, clinically expeditious, cost-effective approach. We evaluated its potential in a well-established, critical-size, rat femoral defect model. METHODS: A critical-size defect was created in the right femur of forty-eight skeletally mature Sprague-Dawley rats. After twenty-four hours, each defect received a single, intralesional, percutaneous injection of adenovirus carrying bone morphogenetic protein-2 (Ad.BMP-2) or luciferase cDNA (Ad.luc) or it remained untreated. Healing was monitored with weekly radiographs. At eight weeks, the rats were killed and the femora were evaluated with dual-energy x-ray absorptiometry, micro-computed tomography, histological analysis, histomorphometry, and torsional mechanical testing. RESULTS: Radiographically, 75% of the Ad.BMP-2-treated femora showed osseous union. Bone mineral content was similar between the Ad.BMP-2-treated femora (0.045 +/- 0.020 g) and the contralateral, intact femora (0.047 +/- 0.003 g). Histologically, 50% of the Ad.BMP-2-treated defects were bridged by lamellar, trabecular bone; the other 50% contained islands of cartilage. The control (Ad.luc-treated) defects were filled with fibrous tissue. Histomorphometry demonstrated a large difference in osteogenesis between the Ad.BMP-2 group (mean bone area, 3.25 +/- 0.67 mm(2)) and the controls (mean bone area, 0.65 +/- 0.67 mm(2)). By eight weeks, the Ad.BMP-2-treated femora had approximately one-fourth of the strength (mean, 0.07 +/- 0.04 Nm) and stiffness (mean, 0.5 +/- 0.4 Nm/rad) of the contralateral femora (0.3 +/- 0.08 Nm and 2.0 +/- 0.5 Nm/rad, respectively). CONCLUSIONS: A single, percutaneous, intralesional injection of Ad.BMP-2 induces healing of critical-size femoral bone defects in rats within eight weeks. At this time, the repair tissue is predominantly trabecular bone, has normal bone mineral content, and has gained mechanical strength.     

A temporary decrease in mineral density in perinatal mouse long bones

Fetal and postnatal bone development in humans is traditionally viewed as a process characterized by progressively increasing mineral density. Yet, a temporary decrease in mineral density has been described in the long bones of infants in the immediate postnatal period. The mechanism that underlies this phenomenon, as well as its causes and consequences, remain unclear. Using daily μCT scans of murine femora and tibiae during perinatal development, we show that a temporary decrease in tissue mineral density (TMD) is evident in mice. By monitoring spatial and temporal structural changes during normal growth and in a mouse strain in which osteoclasts are non-functional (Src-null), we show that endosteal bone resorption is the main cause for the perinatal decrease in TMD. Mechanical testing revealed that this temporary decrease is correlated with reduced stiffness of the bones. We also show, by administration of a progestational agent to pregnant mice, that the decrease in TMD is not the result of parturition itself. This study provides a comprehensive view of perinatal long bone development in mice, and describes the process as well as the consequences of density fluctuation during this period.     

Temporal changes in bone mass and mechanical properties in a murine model of tumor osteolysis

Pathological fracture is a devastating complication of osteolytic bone metastases. The progression of osteolysis and its effect on bone fracture risk are poorly understood. The goal of this study was to determine the temporal changes in bone strength following tumor inoculation in a preclinical model of tumor osteolysis. In addition, a predictive model was developed between non-invasive radiographic measures and bone strength. The right femora of female nude mice were injected with breast cancer cells; the left limb served as a sham-operated control. Radiographs and DEXA scans were obtained at the time of surgery and at 3, 6, and 9 weeks. Groups of mice were euthanized at each time point for mechanical assessment. Micro-CT analysis was performed on a sub-set of mice with advanced state disease to quantify bone loss. Radiographs documented an increase in tumor osteolysis over time, with 58% of the mice showing signs of osteolysis at 3 weeks, 75% at 6 weeks, and 81% at 9 weeks. BMD measurements revealed a 21.6% increase from baseline in the controls whereas tumor-injected femora failed to increase in BMD over the same time course. Tumor-bearing limbs exhibited statistically significant decreases in torque at failure (86%), energy to failure (88%), and initial stiffness (94%) compared to the controls. Both lysis scores and BMD measurements proved to be modest predictors of mechanical strength, accounting for approximately 73% and 41% of variation in torque at failure, respectively. Micro-CT analysis revealed decreases in both total bone volume in the distal femur (31%) and metaphyseal fractional trabecular bone (89%). We have shown that non-invasive radiographic techniques provide a useful tool for monitoring the progression of tumor osteolysis and for predicting the mechanical strength of tumor-bearing bones in this model. By integrating non-invasive measures of tumor osteolysis and fracture risk, we have validated a clinically relevant platform for evaluating new therapeutic approaches for preserving and/or restoring bone affected by metastatic disease.     

Ibandronate treatment decreases the effects of tumor-associated lesions on bone density and strength in the rat.

Bisphosphonate treatment is beneficial against symptoms of metastatic bone disease, although less is known about the effect of preventative treatment schedules. We investigated the effect of various treatment regimens of the bisphosphonate, ibandronate (IB), on the preservation of bone quality in a rat model of tumor-induced osteolysis. Osteolytic Walker 256 (W256) carcinosarcoma cells were implanted into the left femur of female Sprague-Dawley rats, resulting in a 10% reduction in bone mineral density (BMD), a 16% reduction in bone density (BD), and a 26% reduction in failure load compared with the right femur 28 days after implantation. IB was administered subcutaneously in five different treatment schedules: (1) IB PRE-POST received IB for 26 days, prior to implantation of W256 cells in the medullary canal of the femur, and for 28 additional days after surgery; (2) IB PRE-POST SHAM received the same IB administration, but with a sham operation; (3) IB PRE received IB injections before W256 cell insertion only; (4) IB PRE-0 received IB injections for 26 days and was then killed to serve as a time zero control; and (5) IB POST received sham injection with saline before W256 cell insertion, and then received IB injections for 28 days until killing. Controls (TUMOR ONLY) received sham injections with saline prior to W256 cell insertion, and then for 28 additional days until killing. We used dual-energy X-ray absorptiometry (DXA) to measure distal femur BMD and bone mineral content (BMC), peripheral quantitative computed tomography (pQCT) to measure distal femur BD, and torsion testing to obtain torsional failure load. Combined preventative and interventional IB treatment best preserved bone mass and strength, although all treatment schedules resulted in significant improvement compared with untreated controls (TUMOR ONLY). The possibility of reducing or even preventing skeletal morbidity in cancer patients with a high risk of developing metastatic spreading to bone is exciting, and warrants further exploration.     

Mechanical effects of the extended trochanteric osteotomy

BACKGROUND: The extended trochanteric osteotomy was introduced as a safe and effective exposure technique for revision hip surgery; however, intraoperative iatrogenic femoral fractures have been reported. This study examined the effects of the extended trochanteric osteotomy on the torsional strength of the femur with use of cadaver bones. We hypothesized that repair of the osteotomy fragment would restore the torsional strength to that of an intact femur and that an osteotomized femur containing a well-fixed stem would have the same torsional strength as an intact femur with a stem. METHODS: Fifty-eight cadaveric human femora were divided into five groups, according to the repair technique, to examine the effects of the extended trochanteric osteotomy: intact, osteotomy, repaired osteotomy, implant, and implant-repaired osteotomy. Osteotomy fragments were reattached with use of three double-looped 18-gauge wires. A femoral stem was cemented into the last two groups. Specimens were tested mechanically in rotation until failure. Rotational properties were compared with one-way analysis of variance followed by post hoc pairwise comparisons. Linear regression analysis was performed for bone mineral density and torsional strength. RESULTS: Torque to failure was reduced by 73% for the specimens in the osteotomy group compared with the intact group (p < 0.0001). Repair of the osteotomy did not improve torque to failure (p > 0.99). Femora in the implant-repaired osteotomy group displayed significantly improved torque-to-failure values compared with the specimens in the osteotomy and repaired osteotomy groups (p < 0.0001). However, the strength of the femora in the implant-repaired osteotomy group remained significantly less than that of the specimens in the implant group (p < 0.007). A significant linear relationship was observed between bone mineral density and torque to failure for femora in the intact (p < 0.006), osteotomy (p < 0.002), and repaired osteotomy (p < 0.001) groups. CONCLUSIONS: The extended trochanteric osteotomy reduces torsional strength by 73% even when the osteotomy fragment is repaired. Bone mineral density directly affects absolute femoral strength in this model.     

Torsional strength estimates of femoral diaphyses with endosteal lytic lesions: dual-energy X-ray absorptiometry study.

Pathologic fracture is a significant problem for individuals with metastatic bone disease. Current guidelines for prophylactic internal fixation are neither reliable nor easily applied. The purpose of this study was to validate dual-energy X-ray absorptiometry (DXA) as an accurate method for estimating torsional bone strength of diaphyseal bone with endosteal lytic lesions. Endosteal lesions of varying sizes were simulated in the diaphyses of 12 adult cadaveric femurs. Unaltered contralateral femurs served as matched controls. Machined lesions ranged from 3 to 6.5 cm in length, 1 to 3 cm in width, 15 to 48 cm(2) in elliptical area, with 10% to 100% removal of the cortical thickness. Morphology and density data obtained from DXA images were used to estimate torsional strength. All femora were mechanically tested to failure in torsion. Physically measured torsional strength was not significantly correlated to lesion elliptical area (r = 0.542, p > 0.05) or percentage cortical thickness removed (r = 0.257, p > 0.05). Measured torsional strength was significantly correlated to DXA-based torsional strength estimates (r = 0.855, p < 0.01). Lesion size alone did not correlate with the strength of bones with simulated endosteal lytic lesions. In contrast, calculations based on DXA (morphology, density) did correlate with torsional strength. This is the first step in the development of a DXA-based tool for objectively estimating bone strength in the presence of endosteal lytic lesions.     

Supraphysiologic levels of testosterone affect cancellous and cortical bone in the young female Cynomolgus monkey

The goal of this study was to evaluate the effects of chronically-elevated male levels of the potent androgen testosterone on the quality and quantity of both cancellous and cortical bone in a young (mean age 8.0 years), nonhuman female primate model (M. fascicularis). Thirteen intact female monkeys received continuous testosterone supplementation via subcutaneous implants over a 24-month period. A group of 16 untreated, intact, age-matched female monkeys served as controls. At sacrifice, the lumbar vertebrae and femora were recovered in order to analyze the bone mineral quality and quantity of cancellous and cortical bone, respectively, and compared to the control group. Mineralization profiles of the vertebrae and femora were obtained using the density fractionation technique. Chemical analysis of the three largest fractions retrieved by density fractionation was performed to evaluate differences in %Ca, %P, Ca/P ratio, and mineral content (%Ca + %PO₄) between the control and experimental groups. In addition, unfractionated bone powder was examined by X-ray diffraction to identify any changes in crystal size. Coronal sections of vertebrae were analyzed for structural parameters using histomorphometry and image analysis. Cross sections taken at the midshaft diaphyseal femora were analyzed for structural macroscopic and intracortical parameters. A nonsignificant shift in the mineralization profile of the vertebrae was observed whereas there was a significant shift in the mineralization profile towards more dense bone in the treated femora as compared with controls (P < 0.05). There was no difference in terms of size/strain of the cortical or cancellous bone crystal as detected by X-ray diffraction. There was a trend towards an increase in cancellous bone area (B.Ar.) in the testosterone-treated vertebrae (P= 0.08) as compared with controls. The architecture of the cancellous bone remained nonsignificantly different between the treatment and control groups as evaluated by image analysis. There was a decrease in osteoid perimeter (P= 0.05) in the experimental group as compared with controls. There was a significant decrease in eroded perimeter measurements in the experimental group as compared with controls (P < 0.03). Although there was a trend towards an increase in cancellous bone area, mineralization was not significantly different in the vertebrae of testosterone-treated female monkeys, indicating that the newly-formed bone tissue became relatively normally mineralized over the two-year period. An increase in bone area, with indices of an overall decreased remodelling pattern as compared with controls, suggests that cancellous bone in the young, nonhuman female primate had been receptive to supraphysiologic levels of testosterone supplementation over the two-year period. There was a trend for an increase in cortical bone area and width with an increased periosteal perimeter in the testosterone-treated group as compare with controls. There was an increase in intracortical remodelling activity with a significant increase in percent porosity (P < 0.05), osteonal bone (P < 0.05), and mean wall width (P < 0.05) in the testosterone-treated group. In conclusion, the cancellous bone from female monkeys appeared to respond to the antiresorptive stimulus of male levels of testosterone with significantly diminished turnover parameters in this compartment. In contrast, the cortical bone compartment responded by displaying significant intracortical remodelling over a two-year period. Received: 22 December 1995 / Accepted: 3 April 1996     

Protection from stress in bone and its effects. Experiments with stainless steel and plastic plates in dogs.

In a preliminary experiment the paired radii and femora of dogs were tested for bone mineral mass and mechanical properties including the load at break, the ultimate bending strength and the modulus of elasticity; symmetry was observed for most of the parameters determined. The influence of the elasticity of materials used for the internal splintage of bone and its relationship to bone remodeling were then investigated for stainless steel and plastic plates applied to the femora of dogs. A significant decrease in bone mineral mass per centimetre length of bone and in mechanical properties was demonstrated for the femora plated with steel, and microradiography showed that this was due to massive endosteal resorption. A model for determining the influence of protection from stress in bone is presented.     

Augmentation of femoral neck fracture fixation with an injectable calcium-phosphate bone mineral cement

The first goal of this study was to determine if augmentation with an injectable, in situ setting, calcium-phosphate cement that is capable of being remodeled and was designed to mimic bone mineral significantly improved the strength and stiffness of fixation in a cadaveric femoral neck fracture model. The second goal was to determine if greater increases in fixation strength were achieved as the bone density of the specimen decreased. Sixteen pairs of fresh cadaveric human femora with a mean age of 70.9 years (SD = 17.2 years) were utilized. The bone density of the femoral neck was measured with dual-energy x-ray absorptiometry. The femoral head was impacted vertically with the femoral shaft fixed in 12° of adduction using a materials testing machine to create a fully displaced fracture. Following fracture, 30% inferior comminution was created in each specimen. One randomly chosen femur from each pair underwent anatomic reduction and fixation with three cannulated cancellous bone screws, 7 mm in diameter, in an inverted triangle configuration. The contralateral femur underwent the same fixation augmented with calcium-phosphate cement. Specimens were preconditioned followed by 1,000 cycles to one body weight (611.6 N) at 0.5 Hz to simulate single-limb stance loading. The stiffness in the first cycle was observed to be significantly greater in cement-augmented specimens compared with unaugmented controls (p < 0.05). After cycling, each specimen was loaded at 10 mm/min until complete displacement of the fracture surface and failure of fixation occurred. Specimens augmented with bone mineral cement failed at a mean of 4,573 N (SD = 1,243 N); this was significantly greater (p < 0.01) than the mean for controls (3,092 N, SD = 1,258 N). The relative improvement in fixation strength (augmented/control × 100% ) was not inversely correlated to femoral neck bone density (p = 0.25, R² = 0.09), was weakly correlated to the volume of cement injected (p = 0.07, R² = 0.22), and was inversely related to the fixation failure load of the control specimen (p = 0.001, R² = 0.54). There was a mean relative improvement in fixation strength of 169.6% (SD = 77.5). These findings suggest that calcium-phosphate cement provides initial beneficial augmentation to fixation of femoral neck fractures.     

Zoledronic acid decreased osteolysis but not bone metastasis in a nude mouse model of canine prostate cancer with mixed bone lesions

BACKGROUND: Bone metastasis is the most common cause of morbidity and mortality in patients with advanced prostate cancer and is manifested primarily as mixed osteoblastic and osteolytic lesions. However, the mechanisms responsible for bone metastases in prostate cancer are not clearly understood, in part due to the lack of relevant in vivo models that mimic the clinical presentation of the disease in humans. We previously established a nude mouse model with mixed bone metastases using intracardiac injection of canine prostate cancer cells (Ace-1). In this study, we hypothesized that tumor-induced osteolysis promoted the incidence of bone metastases and osteoblastic activity. METHODS: We studied the effect of inhibition of osteolysis with zoledronic acid (ZA) on the prevention and progression of Ace-1 bone metastases in nude mice using prophylactic and delayed treatment protocols. Bioluminescent imaging, radiography, and histopathological evaluation were performed to monitor the effect of ZA on the incidence, progression and nature of bone metastases. RESULTS: Unexpectedly, there was no significant difference in tumor burden and the incidence of metastasis between control and treatment groups as detected by bioluminescent imaging and bone histomorphometry. However, radiographic and histopathological analysis showed a significant treatment-related decrease in osteolysis, but no effect on tumor-induced trabecular bone thickness in both treatment groups compared to controls. CONCLUSION: Our results demonstrated that the incidence of prostate cancer bone metastases in vivo was not reduced by zoledronic acid even though zoledronic acid inhibited bone resorption and bone loss associated with the mixed osteoblastic/osteolytic bone metastases in the Ace-1 model.     

Transplantation of a vascularized rabbit femoral diaphyseal segment: mechanical and histologic properties of a new living bone transplantation model

A new vascularized bone transplantation model is described, including the anatomy and surgical technique of isolating a rabbit femoral diaphyseal segment on its nutrient vascular pedicle. The histologic and biomechanical parameters of pedicled vascularized femoral autotransplants were studied following orthotopic reimplantation in the resulting mid-diaphyseal defect. Vascularized femur segments were isolated in 10 rabbits on their nutrient pedicle, and then replaced orthotopically with appropriate internal fixation. Postoperative weightbearing and mobility were unrestricted, and the contralateral femora served as no-treatment controls. After 16 weeks, the bone flaps were evaluated by x-ray (bone healing), mechanical testing (material properties), microangiography (quantification of intraosseous vasculature), histology (bone viability), and histomorphometry (bone remodeling). Bone healing occurred by 2 weeks, with further callus remodeling throughout the survival period. Eight transplants healed completely, while two had a distal pseudarthrosis. Microangiography demonstrated patent pedicles in all transplants. Intraosseous vessel densities were comparable to nonoperated (control) femora. We found ultimate strength and elastic modulus to be significantly reduced when compared to normal controls. Viable bone, increased mineral apposition rate, and bone turnover were demonstrated in all transplants. The method described, and the data provided will be of value for the further study of isolated segments of living bone, and in particular, for investigations of reconstruction of segmental bone loss in weight-bearing animal models. This study also provides important normative data on living autologous bone flap material properties, vascularity, and bone remodeling. We intend to use this method and data for comparison in subsequent studies of large bone vascularized allotransplantation.     

Experimental changes in mineral content of juvenile mouse femora

Summary Juvenile laboratory mice were exposed to hypergravity (8 g), burrowing exercise, swimming to exhaustion, an anabolic steroid, and swimming and an anabolic steroid for 30 days to determine the variability of skeletal mineralization during growth. Changes in mineralization were correlated with changes in bending strength. Experimental mouse femora were loaded to failure in a cantilever beam configuration to determine bending strength, and ashed to determine total mineral content. Between experimental groups, mineral content ranged from 66.0 to 71.2% with the greatest change from the control being a 4.7% decrease in mineralization in the male swimming exercise group (P<0.001). Within two age-matched experiments, the first showed that the group with the greatest decrease in mineralization also had the greatest reduction in bending strength (P<0.001). The second age-matched experiment showed that the group with the greatest reduction in mineralization had bending strength greatly reduced (P<0.001). However, in this experiment, the weakest femora were in the anabolic steroid group that did not have the mineral content reduced. We conclude that (1) mineralization of juvenile mouse femora is extremely variable given varied conditions of exercise or loading; (2) mineralization of normal bone is decreased more often and to a greater extent than increased from normal exercise controls; (3) the decrease in mineralization seen here can decrease bending strength; and (4) the decrease in mineralization seen was not caused by a decrease in mechanical loading but was probably due to a corticosterone-mediated psychological stress response. Therefore, two variables were indicated, one anabolic (mechanical loading) and one catabolic (corticosterone).     

Effect of simulated metastatic lesions on the biomechanical behavior of the proximal femur

Pathologic fractures of femora in patients with metastatic cancer are associated with high morbidity and mortality. Prediction of impending fractures is based on unspecific clinical criteria or past clinician's experience, which leads to underestimation or overtreatment. The aim of this study was to investigate the effect of the site of metastatic lesions on biomechanical behavior of the proximal femur. Sixteen pairs of human femora were scanned with quantitative computed tomography (QCT) to asses bone mineral density. One femur of each pair remained intact while a defined lesion was reamed out in either the superolateral or inferomedial portion of the femoral neck of the contralateral femur. All femora were loaded in a mechanical test setup mimicking one‐legged stance and stiffness, failure load, and fracture location were determined. In the biomechanical experiments the superolateral lesion and the inferomedial lesion caused a stiffness reduction of 19% and 66%, respectively. The average failure load was 40% and 75% lower for specimens with the superolateral (4.53 ± 1.56 kN) and inferomedial (1.89 ± 1.73 kN) lesions, respectively, compared to intact specimens (7.66 ± 3.34 kN). Lesions in the femoral neck led to reduction in both stiffness and failure load of the proximal femur. Furthermore, the site of the lesion had a large effect on the magnitude of the reduction in biomechanical properties. The presented data emphasize the importance of differentiating between locations of the lesion in pathologic fracture prediction of the metastatic femur and underline the insufficient accuracy of current predictive guidelines. © 2017 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:2407–2414, 2017.

     

The interindividual variation in femoral neck width is associated with the acquisition of predictable sets of morphological and tissue-quality traits and differential bone loss patterns

A better understanding of femoral neck structure and age-related bone loss will benefit research aimed at reducing fracture risk. We used the natural variation in robustness (bone width relative to length) to analyze how adaptive processes covary traits in association with robustness, and whether the variation in robustness affects age-related bone loss patterns. Femoral necks from 49 female cadavers (29-93 years of age) were evaluated for morphological and tissue-level traits using radiography, peripheral quantitative computed tomography, micro-computed tomography, and ash-content analysis. Femoral neck robustness was normally distributed and varied widely with a coefficient of variation of 14.9%. Age-adjusted partial regression analysis revealed significant negative correlations (p < 0.05) between robustness and relative cortical area, cortical tissue-mineral density (Ct.TMD), and trabecular bone mineral density (Ma.BMD). Path analysis confirmed these results showing that a one standard deviation (SD) increase in robustness was associated with a 0.70 SD decrease in RCA, 0.47 SD decrease in Ct.TMD, and 0.43 SD decrease in Ma.BMD. Significantly different bone loss patterns were observed when comparing the most slender and most robust tertiles. Robust femora showed significant negative correlations with age for cortical area (R(2) = 0.29, p < 0.03), Ma.BMD (R(2) = 0.34, p < 0.01), and Ct.TMD (R(2) = 0.4, p < 0.003). However, slender femora did not show these age-related changes (R(2) < 0.09, p > 0.2). The results indicated that slender femora were constructed with a different set of traits compared to robust femora, and that the natural variation in robustness was a determinant of age-related bone loss patterns. Clinical diagnoses and treatments may benefit from a better understanding of these robustness-specific structural and aging patterns.     

Zoledronic acid prevents osteopenia and increases bone strength in a rabbit model of distraction osteogenesis.

Prolonged healing times and stress-shielding osteopenia remain problematic in distraction osteogenesis. In this study of 30 rabbits, zoledronic acid increased regenerate volume, mineralization, and tibial strength and prevented osteopenia over a 6-week period. Translation to the clinical setting, if safe, could improve outcomes in distraction osteogenesis in children. INTRODUCTION: Because the external fixators for limb lengthening and reconstruction are designed to control the positions of bone fragments accurately, they also produce stress-shielding effects on the forming regenerate and surrounding bone. Osteopenia, leading to refracture and limitations on rehabilitation, are common consequences, potentially increasing morbidity and detracting from final clinical outcome. MATERIALS AND METHODS: We examined the effect of zoledronic acid on distraction osteogenesis in 42 immature male NZW rabbits. The model chosen results in reliable regenerate formation and stress-shielding osteopenia. Fourteen animals received either Saline, zoledronic acid 0.1 mg/kg at surgery (ZOL), or another dose 2 weeks postoperatively (Redosed ZOL). Rabbits underwent DXA for bone mineral content and bone mineral density in regenerate and surrounding segments of operated and contralateral tibias. After death at 6 weeks, 30 pairs of tibias underwent quantitative computerized tomography (QCT) and four-point bend testing, and 12 were examined by histomorphometry. The study was powered at 0.8 to show differences of 1.3 SDs for mineral and mechanical parameters. RESULTS: Osteopenia observed in tibias of the Saline group was absent in ZOL and Redosed ZOL tibias, the latter exhibiting higher bone mineral density and bone mineral content over contralateral regions (p < 0.01). Regenerate bone mineral content was higher in ZOL and Redosed ZOL versus Saline groups at 4 and 6 weeks (p < 0.01). Cross-sectional area was 49% and 59% greater at 6 weeks in ZOL and Redosed ZOL regenerates compared with the Saline group (p < 0.01). ZOL and Redosed ZOL tibias were 29% and 89% stronger by four-point bending than the Saline group (p < 0.01). Histomorphometry in the regenerate of ZOL and Redosed ZOL groups revealed higher trabecular bone volume and trabecular number compared with the Saline group (p < 0.001). CONCLUSIONS: Zoledronic acid administration led to significantly greater bone area, mineral content, strength, and trabecular number with reduced stress-shielding osteopenia in this model of distraction osteogenesis. These data suggest that intraoperative and postoperative zoledronic acid administration could improve outcomes in children undergoing limb lengthening.     

Effect of long-term impact-loading on mass, size, and estimated strength of humerus and radius of female racquet-sports players: a peripheral quantitative computed tomography study between young and old starters and controls.

Bone characteristics of the humeral shaft and distal radius were measured from 64 female tennis and squash players and their 27 age-, height-, and weight-matched controls with peripheral quantitative tomography (pQCT) and dual energy X-ray absorptiometry (DXA). The players were divided into two groups according to the starting age of their tennis or squash training (either before or after menarche) to examine the possible differences in the loading-induced changes in bone structure and volumetric density. The following pQCT variables were used: bone mineral content, total cross-sectional area of bone (TotA), cross-sectional area of the marrow cavity (CavA) and that of the cortical bone (CoA), cortical wall thickness (CWT), volumetric density of the cortical bone (CoD) and trabecular bone (TrD), and torsional bone strength index for the shaft (BSIt) and compressional bone strength index for the bone end (BSIc). These bone strength indices were compared with the DXA-derived areal bone mineral density (aBMD) to assess how well the latter represents the effect of mechanical loading on apparent bone strength. At the humeral shaft, the loaded arm's greater bone mineral content (an average 19% side-to-side difference in young starters and 9% in old starters), was caused by an enlarged cortex (CoA; side-to-side differences 20% and 9%, respectively). The loaded humerus seemed to have grown periosteally (the CavA did not differ between the sites), leading to 26% and 11% side-to-side BSIt differences in the young and old starters, respectively. CoD was equal between the arms (-1% difference in both player groups). The side-to-side differences in the young starters' bone mineral content, CoA, TotA, CWT, and BSIt were 8-22% higher than those of the controls and 8-14% higher than those of the old starters. Old starters' bone mineral content, CoA, and BSIt side-to-side differences were 6-7% greater than those in the controls. The DXA-derived side-to-side aBMD difference was 7% greater in young starters compared with that of the old starters and 14% compared with that in controls, whereas the difference between old starters and controls was 6%, in favor of the former. All these between-group differences were statistically significant. At the distal radius, the player groups differed significantly from controls in the side-to-side bone mineral content, TrD, and aBMD differences only: the young starters' bone mineral content difference was 9% greater, TrD and aBMD differences were 5% greater than those in the controls, and the old starters' TrD and aBMD differences were both 7% greater than those in the controls. In summary, in both of the female player groups, the structural adaptation of the humeral shaft to long-term loading seemed to be achieved through periosteal enlargement of the bone cortex, although this adaptation was clearly better in the young starters. Exercise-induced cortical enlargement was not so clear at the distal radius (a trabecular bone site), and the study suggested that at long bone ends, the trabecular density could be a modifiable factor to built a stronger bone structure. Conventional DXA-based aBMD measurement detected the intergroup differences in the exercise-induced bone gains, although, because it measured two dimensions of bone only, it seemed to underestimate the effect of exercise on the apparent bone strength, especially if the playing had been started during the growing years.     

Leptin deficiency produces contrasting phenotypes in bones of the limb and spine

Leptin is a hormone secreted by adipocytes that can regulate bone mass through a central, neuroendocrine signaling pathway. We tested the hypothesis that the response of bone tissue to altered leptin signaling is not uniform throughout the skeleton, but may vary between different skeletal regions and between cortical and trabecular moieties. We investigated the effects of leptin deficiency on muscle mass and bone architecture in obese, leptin-deficient (ob/ob) mice, and in lean controls. Results indicate that the obese mice weigh approximately twice as much as the lean mice, but the quadriceps muscles of the ob/ob mice are 40% smaller than those of controls. Leptin-deficient mice have significantly shorter femora, lower femoral bone mineral content (BMC), bone mineral density (BMD), cortical thickness, and trabecular bone volume compared to lean mice. Marrow tissue from the femora of ob/ob mice also shows a marked increase in adipocyte number compared to that of normal mice. In contrast to the pattern observed in the femur, ob/ob mice have significantly increased vertebral length, lumbar BMC, lumbar BMD, and trabecular bone volume compared to lean controls. Few adipocytes are observed in bone marrow from lumbar vertebrae of ob/ob mice, despite being numerous in marrow of the femur. However, like the femur, significant cortical thinning is also observed in the spine. These results indicate that the effects of altered leptin signaling on bone differ significantly between axial and appendicular regions, and may be mediated in part by muscle mass. The muscle hypoplasia, increased marrow adipogenesis, and decreased bone mass observed in the hindlimbs of ob/ob mice are also observed with aging in humans, suggesting that the ob/ob mouse may be a new and useful animal model for studying the relationship between bone marrow adipogenesis and osteopenia.     

A quantitative evaluation of periprosthetic bone-remodeling after cementless total hip arthroplasty.

Dual-energy x-ray absorptiometry analysis was used to determine the periprosthetic bone-mineral content of ten femora that had been obtained at the autopsies of five elderly patients who had had an AML (anatomic medullary locking) prosthesis in situ for seventeen to eighty-four months. Clinical radiographs showed resorptive remodeling changes characteristic of femora containing this implant. Before the absorptiometry was performed, an identical prosthesis had been inserted into the contralateral, normal femur of each cadaver. The mean difference in the periprosthetic bone-mineral content between the remodeled femora and the femora in which the prosthesis had been implanted post mortem ranged from 7 to 52 per cent, with the bone-mineral content always less in the remodeled femora. The greatest mean decrease in bone-mineral content (45 per cent) occurred adjacent to the proximal one-third of the remodeled femora. The percentage decrease in periprosthetic bone-mineral content in the remodeled femora had an inverse linear relationship with the corresponding bone-mineral content of the contralateral control femora. Preoperative analysis of bone density may therefore be useful for prediction of the extent of resorptive bone-remodeling after total hip replacement.     

The effect of growth/differentiation factor-5 deficiency on femoral composition and mechanical behavior in mice

A subclass of the bone morphogenetic proteins (BMPs), known as growth/differentiation factors (GDFs) 5, 6, and 7, have been shown to affect several skeletal processes, including endochondral ossification, synovial joint formation, and tendon and ligament repair. Mice deficient in GDF-5 have also been shown to exhibit biomechanical abnormalities in tendon that may be associated with altered type I collagen. The purpose of this study was to investigate the effect of GDF-5 deficiency on another type I collagen-rich tissue: cortical bone. Analyses were performed on femora from 8-week-old GDF-5-deficient male brachypodism mice. We hypothesized that GDF-5-deficient bones would exhibit altered geometric, structural, and material properties compared with control littermates. Mutant animals were significantly smaller in body mass than controls (-21%). Geometrically, mutant long bones were significantly shorter (-25%), had a lower polar moment of inertia (-34%), and a lower geometric strength indicator (analogous to the section modulus of a circular section) (-30%). When normalized by body mass, however, geometric differences were no longer significant. Structurally, GDF-5-deficient femora were weaker (-31%) and more compliant (-57%) than controls when tested to failure in torsion. Lower bone structural stiffness in the mutants was not completely explained by the smaller bone geometry, because mutant bones exhibited a significantly lower effective shear modulus (-36%). Although body mass did not fully explain the reduced structural strength in mutant bones, strength differences were adequately explained by bone cross-sectional geometry; maximum effective shear stress was not significantly different between mutants and controls, despite a statistically significant 6% lower ash fraction in mutant femora. No significant difference was detected in collagen content, as indicated by hydroxyproline per dry mass.     

Remineralization of lytic spinal metastases after radiotherapy

BACKGROUND CONTEXTPalliative radiotherapy (RT) can lead to remineralization of osteolytic lesions thereby potentially restoring some of the weight-bearing capacity and preventing vertebral collapse. It is not clear, however, under which circumstances remineralization of osteolytic lesions occurs.PURPOSEThe aim of this study was to investigate the change in bone mineral density in spinal metastases after RT compared to a reference region, and find associated factors.STUDY DESIGNRetrospective analysis within prospective observational cohortOUTCOME MEASURESchange in bone mineral density measured in Hounsfield Units (HU).PATIENT SAMPLEpatients treated with RT for (painful) bone metastases.METHODSPatients with spinal metastases were included if computed tomography scans both pre- and post-RT were available. Bone density was measured in HU. A region of interest (ROI) was drawn manually in the metastatic lesion. As a reference, a measurement of bone density in adjacent, unaffected, and non-irradiated vertebrae was used. Factors tested for association were origin of the primary tumor, RT dose and fractionation scheme, and concomitant use of bisphosphonates.RESULTSA total of 31 patients with 49 spinal metastases, originating from various primary tumors, were included. The median age on baseline was 58 years (IQR: 53–63) and median time between baseline and follow-up scan was 8.2 months (IQR: 3.0–18.4). Difference in HU in the lesion before and after treatment was 146.9 HU (95% CI 68.4–225.4; p<.01). Difference in HU in the reference vertebra between baseline and first follow-up was 19.1 HU (95% CI -47.9 to 86.0; p=.58). Difference between reference vertebrae and metastatic lesions on baseline was -194.1 HU (95% CI -276.2 to -112.0; p<.01). After RT, this difference was reduced to -50.3 HU (95% CI -199.6 to 99.0; p=.52). Patients using bisphosphonates showed a greater increase in HU, 194.1 HU versus 60.6 HU, p=.01.CONCLUSIONSPalliative radiation of osteolytic lytic spinal metastases is positively associated with an increased bone mineral density at follow-up. The use of bisphosphonates was linked to an increased bone mineral density when used during or after RT.