Angiogenesis is required for stress fracture healing in rats

Although angiogenesis and osteogenesis are critically linked, the importance of angiogenesis for stress fracture healing is unknown. In this study, mechanical loading was used to create a non-displaced stress fracture in the adult rat forelimb. Fumagillin, an anti-angiogenic agent, was used as the water soluble analogue TNP-470 (25 mg/kg) as well as incorporated into lipid-encapsulated α_vβ₃ integrin targeted nanoparticles (0.25 mg/kg). In the first experiment, TNP-470 was administered daily for 5 days following mechanical loading, and changes in gene expression, vascularity, and woven bone formation were quantified. Although no changes in vascularity were detected 3 days after loading, treatment-related downregulation of angiogenic (Pecam1) and osteogenic (Bsp, Osx) genes was observed at this early time point. On day 7, microCT imaging of loaded limbs revealed diminished woven bone formation in treated limbs compared to vehicle treated limbs. In the second experiment, α_vβ₃ integrin targeted fumagillin nanoparticles were administered as before, albeit with a 100-fold lower dose, and changes in vascularity and woven bone formation were determined. There were no treatment-related changes in vessel count or volume 3 days after loading, although fewer angiogenic (CD105 positive) blood vessels were present in treated limbs compared to vehicle treated limbs. This result manifested on day 7 as a reduction in total vascularity, as measured by histology (vessel count) and microCT (vessel volume). Similar to the first experiment, treated limbs had diminished woven bone formation on day 7 compared to vehicle treated limbs. These results indicate that angiogenesis is required for stress fracture healing, and may have implications for inducing rapid repair of stress fractures.     

Sclerostin antibody treatment improves fracture outcomes in a Type I diabetic mouse model

Type 1 diabetes mellitus (T1DM) patients have osteopenia and impaired fracture healing due to decreased osteoblast activity. Further, no adequate treatments are currently available that can restore impaired healing in T1DM; hence a significant need exists to investigate new therapeutics for treatment of orthopedic complications. Sclerostin (SOST), a WNT antagonist, negatively regulates bone formation, and SostAb is a potent bone anabolic agent. To determine whether SOST antibody (SostAb) treatment improves fracture healing in streptozotocin (STZ) induced T1DM mice, we administered SostAb twice weekly for up to 21 days post-fracture, and examined bone quality and callus outcomes at 21 days and 42 days post-fracture (11 and 14 weeks of age, respectively). Here we show that SostAb treatment improves bone parameters; these improvements persist after cessation of antibody treatment. Markers of osteoblast differentiation such as Runx2, collagen I, osteocalcin, and DMP1 were reduced, while an abundant number of SP7/osterix-positive early osteoblasts were observed on the bone surface of STZ calluses. These results suggest that STZ calluses have poor osteogenesis resulting from failure of osteoblasts to fully differentiate and produce mineralized matrix, which produces a less mineralized callus. SostAb treatment enhanced fracture healing in both normal and STZ groups, and in STZ + SostAb mice, also reversed the lower mineralization seen in STZ calluses. Micro-CT analysis of calluses revealed improved bone parameters with SostAb treatment, and the mineralized bone was comparable to Controls. Additionally, we found sclerostin levels to be elevated in STZ mice and β-catenin activity to be reduced. Consistent with its function as a WNT antagonist, SostAb treatment enhanced β-catenin activity, but also increased the levels of SOST in the callus and in circulation. Our results indicate that SostAb treatment rescues the impaired osteogenesis seen in the STZ induced T1DM fracture model by facilitating osteoblast differentiation and mineralization of bone.     

Difference in the trajectory of change in bone geometry as measured by hip structural analysis in the narrow neck,intertrochanteric region,and femoral shaft between men and women following hip fracture

Prior studies have shown that women have declines in bone structure and strength after hip fracture, but it is unclear whether men sustain similar changes. Therefore, the objective was to examine sex differences in proximal femur geometry following hip fracture. Hip structural analysis was used to derive metrics of bone structure and strength: aerial bone mineral density, cross-sectional bone area (CSA), cortical outer diameter, section modulus (SM), and buckling ratio (BR) from dual-energy x-ray absorptiometry scans performed at baseline (within 22 days of hospital admission), two, six, or twelve months after hip fracture in men and women (n = 282) enrolled in the Baltimore Hip Studies 7th cohort. Weighted estimating equations were used to evaluate sex differences at the narrow neck (NN), intertrochanteric (IT), and femoral shaft (FS). Men had significantly different one year NN changes compared to women in CSA: − 6.33% (− 12.47, − 0.20) vs. 1.37% (− 3.31, 6.43), P = 0.049; SM: − 4.98% (− 11.08, 1.10) vs. 3.94% (− 2.51, 10.42), P = 0.042; and BR: 7.50% (0.65, 14.36) vs. − 1.20% (− 6.41, 4.00), P = 0.044. One year IT changes displayed similar patterns, but the sex differences were not statistically significant for CSA: − 4.07% (− 10.83, 2.67) vs. 0.41% (− 3.41, 4.24), P = 0.252; SM: − 4.78% (− 12.10, 5.53) vs. -0.31 (− 4.74, 4.11), P = 0.287; and BR: 4.59% (− 0.65, 9.84) vs. 1.52% (− 4.23, 7.28), P = 0.425. Differences in FS geometric parameters were even smaller in magnitude and not significantly different by sex. Women generally experienced non-significant increases in bone tissue and strength following hip fracture, while men had structural declines that were statistically greater at the NN region. Reductions in the mechanical strength of the proximal femur after hip fracture could put men at higher risk for subsequent fractures of the contralateral hip.     

Zoledronic acid suppresses callus remodeling but enhances callus strength in an osteoporotic rat model of fracture healing

Mini-abstractIn this study, we demonstrated that the use of zoledronic acid does not impair fracture healing, but results in superior callus size and resistance at the fracture site, which could be the consequence of a lower rate of bone turnover due to its anti-catabolic effect.ObjectiveTo investigate the effect of inhibition of bone remodeling by the bisphosphonate, zoledronic acid, on callus properties in an osteoporotic rat model of fracture healing.MethodsOvariectomized (OVX) rats were randomly divided into four treatment groups (n = 24 per group): saline control (CNT); and three systemic zoledronic acid-injected groups (0.1 mg/kg), administered 1 day (ZOLD1), 1 week (ZOLW1), and 2 weeks (ZOLW2) after fracture. Rats were killed at either 6 or 12 weeks postoperatively. Postmortem analyses included radiography, microcomputed tomography, histology, histomorphometry, biomechanical tests, and nanoindentation tests.ResultsTreatment with zoledronic acid led to a significant increase in trabecular bone volume within the callus, as well as in callus resistance, compared to those in the saline control rats; delayed administration (ZOLW2) reduced intrinsic material properties, including ultimate stress and elastic modulus, and microarchitecture parameters, including bone volume/total volume (BV/TV), trabecular thickness (Tb.Th), and connectivity density (Conn.D), compared with ZOLD1 at 12 weeks after surgery. OVX had a negative effect on the progression of endochondral ossification at 6 weeks. Zoledronic acid administration at an early stage following fracture may bind to early callus, and thus not affect subsequent callus formation and endochondral ossification, while delayed administration (ZOLW2) mildly suppresses bony callus remodeling.ConclusionThe superior results obtained with zoledronic acid (ZOLD1, ZOLW1, and ZOLW2) compared to CNT in terms of callus size and resistance could be the consequence of a lower rate of bone turnover at the fracture site due to the anti-catabolic effect of zoledronic acid. Mild suppression of callus remodeling by delayed administration did not impair the initial phase of the fracture healing process.     

Disruption of glucocorticoid signaling in chondrocytes delays metaphyseal fracture healing but does not affect normal cartilage and bone development

States of glucocorticoid excess are associated with defects in chondrocyte function. Most prominently there is a reduction in linear growth but delayed healing of fractures that require endochondral ossification to also occur. In contrast, little is known about the role of endogenous glucocorticoids in chondrocyte function. As glucocorticoids exert their cellular actions through the glucocorticoid receptor (GR), we aimed to elucidate the role of endogenous glucocorticoids in chondrocyte function in vivo through characterization of tamoxifen-inducible chondrocyte-specific GR knockout (chGRKO) mice in which the GR was deleted at various post-natal ages. Knee joint architecture, cartilage structure, growth plates, intervertebral discs, long bone length and bone micro-architecture were similar in chGRKO and control mice at all ages. Analysis of fracture healing in chGRKO and control mice demonstrated that in metaphyseal fractures, chGRKO mice formed a larger cartilaginous callus at 1 and 2 week post-surgery, as well as a smaller amount of well-mineralized bony callus at the fracture site 4 week post-surgery, when compared to control mice. In contrast, chondrocyte-specific GR knockout did not affect diaphyseal fracture healing. We conclude that endogenous GC signaling in chondrocytes plays an important role during metaphyseal fracture healing but is not essential for normal long bone growth.     

Dynamic acoustic radiation force retains bone structural and mechanical integrity in a functional disuse osteopenia model

Disuse osteopenia and bone loss have been extensively reported in long duration space mission and long term bed rest. The pathology of the bone loss is similar to osteoporosis but highly confined to weight bearing bones. The current anabolic and/or anti-resorptive drugs have systemic effects and are costly over extended time, with concerns of long term fracture risk. This study use Low Intensity Pulsed Ultrasound (LIPUS) as a non-invasive acoustic force and anabolic stimulus to countermeasure disuse induced bone loss. Four-month old C57BL/6 mice were randomized into five groups, 1) age-matched (AM), 2) non-suspended sham (NS), 3) non-suspended-LIPUS (NU), 4) suspended sham (SS), and 5) suspended-LIPUS (SU) groups. After four weeks of suspension, μCT analyses showed significant decreases in trabecular bone volume fraction (BV/TV) (− 36%, p < 0.005), bone tissue mineral density (TMD) (− 3%, p < 0.05), trabecular thickness (Tb.Th) (− 12.5%, p < 0.005), and increase in bone surface/bone volume (+ BS/BV) (+ 16%, p < 0.005), relative to age-matched (AM). The application of LIPUS for 20 min/day for 5 days/week, significantly increased TMD (+ 3%, p < 0.05), Tb.Th (+ 6%, p < 0.05), and decreased BS/BV (− 10%, p < 0.005), relative to suspension alone (SS) mice. Histomorphometry analyses showed a breakdown of bone microstructure under disuse conditions consist with μCT results. In comparison to SS mice, LIPUS treated bone showed increased structural integrity with increased bone formation rates at metaphysical endosteal and trabecular surfaces (+ 0.104 ± 0.07 vs 0.031 ± 0.30 μm³/μm²/day) relative to SS. Four-point bending mechanical tests of disused SS femurs showed reduced elastic modulus (− 53%, p < 0.05), yield (− 33%, p < 0.05) and ultimate strength (− 45%, p < 0.05) at the femoral diaphysis relative to AM bone. LIPUS stimulation mitigated the adverse effects of disuse on bone elastic modulus (+ 42%, p < 0.05), yield strength (+ 29%, p < 0.05), and ultimate strength (+ 39%, p < 0.05) relative to SS femurs. LIPUS provides the essential mechanical stimulus to retain bone morphological and mechanical integrity in disuse conditions. This study demonstrates LIPUS potential as regional therapeutic agent to countermeasure disuse induced bone loss while maintaining bone's integrity.     

Increased fracture callus mineralization and strength in cathepsin K knockout mice

Cathepsin K (CatK) is a cysteine protease, expressed predominantly in osteoclasts (OC) which degrades demineralized bone matrix. Novel selective inhibitors of CatK are currently being developed for the treatment of postmenopausal osteoporosis. Pharmacological inhibition of CatK reduces OC resorption activity while preserving bone formation in preclinical models. Disruption of the CatK gene in mice also results in high bone mass due to impaired bone resorption and elevated formation. Here, we assessed mid-shaft femoral fracture healing in 8–10 week old CatK knock-out (KO) versus wild type (WT) mice. Fracture healing and callus formation were determined in vivo weekly via X-ray, and ex vivo at days 14, 18, 28 and 42 post-fracture by radiographic scoring, micro-computed tomography (μCT), histomorphometry and terminal mechanical four point bend strength testing. Radiological evaluation indicated accelerated bone healing and remodeling for CatK KO animals based on increased total radiographic scores that included callus opacity and bridging at days 28 and 42 post-fracture. Micro-CT based total callus volume was similar in CatK KO and WT mice at day 14. Callus size in CatK KO mice was 25% smaller than that in WT mice at day 18, statistically significant by day 28 and exhibited significantly higher mineralized tissue volume and volumetric BMD as compared to WT by day 18 onward. Osteoclast surface and osteoid surface trended higher in CatK KO calluses at all time-points and osteoblast number was also significantly increased at day 28. Increased CatK KO callus mineral density was reflected in significant increases in peak load and stiffness over WT at day 42 post-fracture. Regression analysis indicated a positive correlation (r = 0.8671; p < 0.001) between callus BMC and peak load indicating normal mineral properties in CatK KO calluses. Taken together, gene deletion of cathepsin K in mice accelerated callus size resolution, significantly increased callus mineralized mass, and improved mechanical strength as compared to wild type mice.     

Bone marrow ablation demonstrates that estrogen plays an important role in osteogenesis and bone turnover via an antioxidative mechanism

To assess the effect of estrogen deficiency on osteogenesis and bone turnover in vivo, 8-week-old mice were sham-operated or bilaterally ovariectomized (OVX), and after 8 weeks, mechanical bone marrow ablation (BMX) was performed and newly formed bone tissue was analyzed from 6 days to 2 weeks after BMX. Our results demonstrated that OVX mice following BMX displayed 2 reversed phase changes, one phase observed at 6 and 8 days after BMX delayed osteogenesis accompanied by a delay in osteoclastogenesis, and the other phase observed at 12 and 14 days after BMX increased osteoblastic activity and osteoclastic activity. Furthermore, we asked whether impaired osteogenesis caused by estrogen deficiency was associated with increased oxidative stress, and oxidative stress parameters were examined in bone tissue from sham-operated and OVX mice and OVX mice were administrated with antioxidant N-acetyl-l-cysteine (NAC) or vehicle after BMX. Results demonstrated that estrogen deficiency induced oxidative stress in mouse bone tissue with reduced antioxidase levels and activity, whereas NAC administration almost rescued the abnormalities in osteogenesis and bone turnover caused by OVX. Results from this study indicate that estrogen deficiency resulted in primarily impaired osteogenesis and subsequently accelerated bone turnover by increasing oxidative stress and oxidative stress promises to be an effective target in the process of treatment of postmenopausal osteoporosis.     

Bone lead (Pb) content at the tibia is associated with thinner distal tibia cortices and lower volumetric bone density in postmenopausal women

Conflicting evidence suggests that bone lead or blood lead may reduce areal bone mineral density (BMD). Little is known about how lead at either compartment affects bone structure. This study examined postmenopausal women (N = 38, mean age 76 ± 8, body mass index (BMI): 26.74 ± 4.26 kg/m²) within the Hamilton cohort of the Canadian Multicentre Osteoporosis Study (CaMos), measuring bone lead at 66% of the non-dominant leg and at the calcaneus using ¹⁰⁹Cadmium X-ray fluorescence. Volumetric BMD and structural parameters were obtained from peripheral quantitative computed tomography images (200 μm in-plane resolution, 2.3 ± 0.5 mm slice thickness) of the same 66% site and of the distal 4% site of the tibia length. Blood lead was measured using atomic absorption spectrometry and blood-to-bone lead partition coefficients (P_BB, log ratio) were computed. Multivariable linear regression examined each of bone lead at the 66% tibia, calcaneus, blood lead and P_BB as related to each of volumetric BMD and structural parameters, adjusting for age and BMI, diabetes or antiresorptive therapy. Regression coefficients were reported along with 95% confidence intervals. Higher amounts of bone lead at the tibia were associated with thinner distal tibia cortices (− 0.972 (− 1.882, − 0.061) per 100 μg Pb/g of bone mineral) and integral volumetric BMD (− 3.05 (− 6.05, − 0.05) per μg Pb/g of bone mineral). A higher P_BB was associated with larger trabecular separation (0.115 (0.053, 0.178)), lower trabecular volumetric BMD (− 26.83 (− 50.37, − 3.29)) and trabecular number (− 0.08 (− 0.14, − 0.02)), per 100 μg Pb/g of bone mineral after adjusting for age and BMI, and remained significant while accounting for diabetes or use of antiresorptives. Total lead exposure activities related to bone lead at the calcaneus (8.29 (0.11, 16.48)) and remained significant after age and antiresorptives-adjustment. Lead accumulated in bone can have a mild insult on bone structure; but greater partitioning of lead in blood versus bone revealed more dramatic effects on both microstructure and volumetric BMD.     

Zoledronate prevents lactation induced bone loss and results in additional post-lactation bone mass in mice

In rodents, lactation is associated with a considerable and very rapid bone loss, which almost completely recovers after weaning. The aim of the present study was to investigate whether the bisphosphonate Zoledronate (Zln) can inhibit lactation induced bone loss, and if Zln interferes with recovery of bone mass after lactation has ceased.Seventy-six 10-weeks-old NMRI mice were divided into the following groups: Baseline, Pregnant, Lactation, Lactation + Zln, Recovery, Recovery + Zln, and Virgin Control (age-matched). The lactation period was 12 days, then the pups were removed, and thereafter recovery took place for 28 days. Zln, 100 μg/kg, was given s.c. on the day of delivery, and again 4 and 8 days later. Mechanical testing, μCT, and dynamic histomorphometry were performed. At L4, lactation resulted in a substantial loss of bone strength (− 55% vs. Pregnant, p < 0.01), BV/TV (− 40% vs. Pregnant, p < 0.01), and trabecular thickness (Tb.Th) (− 29% vs. Pregnant, p < 0.001). Treatment with Zln completely prevented lactation induced loss of bone strength, BV/TV, and Tb.Th at L4. Full recovery of micro-architectural and mechanical properties was found 28 days after weaning in vehicle-treated mice. Interestingly, the recovery group treated with Zln during the lactation period had higher BV/TV (+ 45%, p < 0.01) and Tb.Th (+ 16%, p < 0.05) compared with virgin controls. Similar results were found at the proximal tibia and femur. This indicates that Zln did not interfere with the bone formation taking place after weaning. On this background, we conclude that post-lactation bone formation is not dependent on a preceding lactation induced bone loss.     

A novel method for lateral callus distraction and its importance for the mechano-biology of bone formation

We introduce a novel method of lateral callus distraction for bone formation, which avoids the conventional splitting and weakening of bones. At the medial aspect of the sheep tibia the periosteum was resected and small holes were drilled into the cortex to connect the bone surface with the marrow. A distraction device with a hydroxyapatite-coated titanium plate was fixed over the drilled area. After 10 days latency the plate was distracted perpendicular to the bone's long axis twice a day by 0.27 mm for 10 days. The newly formed tissue was then allowed 50 days of maturation. In a control group the plate was fixed 5.4 mm distant from the bone surface. After 70 days all sheep were sacrificed and investigated histo-morphologically and with pQCT.Significantly more bone had developed between the lateral bone surface and the plate in the distraction group compared to the control group. There was exclusively intra-membranous bone formation with trabeculae oriented in the direction of the applied distraction. The main calcification occurred weeks after the last distraction. In conventional callus distraction the tissue strain caused by distraction is superimposed by the tissue deformation due to the deformation of the fixation device. In contrast, in the newly introduced lateral callus distraction method pure uniaxial strain occurs. From a mechano-biological point of view these results suggest that pure uniaxial strain induces exclusively intra-membranous bone formation. Furthermore, it shows that the anabolic effect of tissue strain is present even 50 days after the last stimulation by distraction.     

Effect of near-infrared light on in vitro cellular ATP production of osteoblasts and fibroblasts and on fracture healing with intramedullary fixation

ObjectiveEvaluate the effect of near-infrared light (NIR) on immediate production of ATP by osteoblasts and fibroblasts in vitro, and the healing process of rat femur fractures with intramedullary fixation.BackgroundNIR is one potential treatment option for complications of fracture healing, which has shown to stimulate cellular proliferation and to enhance the healing process.MethodsCell culture – MC3T3-E1 and 3T3-A31 cells were subjected to NIR at 660 nm, 830 nm, or both combined. ATP was assayed at 5, 10, 20, and 45 min after exposure. Animal study – 18 rats had surgery with retrograde intramedullary pins inserted into their femurs, which then underwent closed, transverse femur fracture. Rats were randomly divided into 3 study groups of 6 each: nonirradiated controls, 660 nm, and 830 nm NIR. Healing process was assessed by a blinded radiologist, assigning a healing score of 1–6 for radiographs taken on days 0, 7, 14, and 21.ResultsCell culture – All groups gave significant increase in ATP within 5–10 min, with decay to baseline by 45 min. 660 nm NIR was significantly more effective than 830 nm with fibroblasts or either wavelength with osteoblasts. Animal study – A significant increase in the fracture healing grade in the 660 nm group at day 14, but with no differences at day 21.ConclusionThe study demonstrated an immediate increase in ATP production in vitro and an initial acceleration of callus formation in the fracture healing process, in the presence of NIR.     

Roles of Wnt/β-catenin signalling pathway in the bony repair of injured growth plate cartilage in young rats

Growth plate cartilage is responsible for longitudinal growth of the long bone in children, and its injury is often repaired by bony tissue, which can cause limb length discrepancy and/or bone angulation deformities. Whilst earlier studies with a rat growth plate injury repair model have identified inflammatory, mesenchymal infiltration, osteogenesis and remodeling responses, the molecular mechanisms involved in the bony repair remain unknown. Since our recent microarray study has strongly suggested involvement of Wnt–β-catenin signalling pathway in regulating the growth plate repair and the pathway is known to play a crucial role in the osteogenic differentiation of mesenchymal progenitor cells, the current study investigated the potential roles of Wnt–β-catenin signalling pathway in the bony repair of injured tibial growth plate in rats. Immunohistochemical analysis of the growth plate injury site revealed β-catenin immunopositive cells within the growth plate injury site. Treatment of the injured rats with the β-catenin inhibitor ICG-001 (oral gavage at 200 mg/kg/day for 8 days, commenced at day 2 post injury) enhanced COL2A1 gene expression (by qRT-PCR) and increased proportion of cartilage tissue (by histological analysis), but decreased level of osterix expression and amount of bone tissue, at the injury site by day 10 post-injury (n = 8, P < 0.01 compared to vehicle controls). Consistently, in vitro studies with bone marrow stromal cells from normal rats showed that β-catenin inhibitor ICG-001 dose dependently inhibited expression of Wnt target genes Cyclin D1 and survivin (P < 0.01). At 25 mM, ICG-001 suppressed osteogenic (by CFU-f-ALP assay) but enhanced chondrogenic (by pellet culture) differentiation. These results suggest that Wnt/β-catenin signalling pathway is involved in regulating growth plate injury repair by promoting osteoblastogenesis, and that intervention of this signalling could represent a potential approach in enhancing cartilage repair after growth plate injury.     

Endochondral fracture healing with external fixation in the Sost knockout mouse results in earlier fibrocartilage callus removal and increased bone volume fraction and strength

Sclerostin deficiency, via genetic knockout or anti-Sclerostin antibody treatment, has been shown to cause increased bone volume, density and strength of calluses following endochondral bone healing. However, there is limited data on the effect of Sclerostin deficiency on the formative early stage of fibrocartilage (non-bony tissue) formation and removal. In this study we extensively investigate the early fibrocartilage callus. Closed tibial fractures were performed on Sost^−/− mice and age-matched wild type (C57Bl/6J) controls and assessed at multiple early time points (7, 10 and 14 days), as well as at 28 days post-fracture after bony union. External fixation was utilized, avoiding internal pinning and minimizing differences in stability stiffness, a variable that has confounded previous research in this area.Normal endochondral ossification progressed in wild type and Sost^−/− mice with equivalent volumes of fibrocartilage formed at early day 7 and day 10 time points, and bony union in both genotypes by day 28. There were no significant differences in rate of bony union; however there were significant increases in fibrocartilage removal from the Sost^−/− fracture calluses at day 14 suggesting earlier progression of endochondral healing. Earlier bone formation was seen in Sost^−/− calluses over wild type with greater bone volume at day 10 (221%, p < 0.01). The resultant Sost^−/− united bony calluses at day 28 had increased bone volume fraction compared to wild type calluses (24%, p < 0.05), and the strength of the fractured Sost^−/− tibiae was greater than that that of wild type fractured tibiae.In summary, bony union was not altered by Sclerostin deficiency in externally-fixed closed tibial fractures, but fibrocartilage removal was enhanced and the resultant united bony calluses had increased bone fraction and increased strength.     

Value of ultrasonography and radiography for the study of bone regeneration in lengthening of the fourth ray in brachymetatarsia

BackgroundDistraction osteogenesis is frequently used for brachymetatarsia.MethodsBrachymetatarsia of the fourth ray was treated with the Ilizarov method in 10 females. Distraction rate was 1.00?0.75 mm a day. Radiography and ultrasonography were used to study the regeneration course throughout the distraction and fixation phases.ResultsMean lengthening achieved was 2.28 cm in the average distraction period of 30.2 days and average fixation phase of 50.2 days. Ultrasonography detected the regeneration signs and zonal structure of the regenerate earlier than radiography. It confirmed slow regeneration in two patients and their distraction rate was corrected. One patient had premature consolidation on distraction day 10 and needed re-osteotomy.ConclusionsCombination of radiography and ultrasonography to study bone regeneration in brachymetatarsisa provides a better monitoring of distraction callus progression. Ultrasonography is of great value to detect regeneration problems during early stages of lengthening.     

Modulation of endochondral ossification by MEK inhibitors PD0325901 and AZD6244 (Selumetinib)

MEK inhibitors (MEKi) PD0325901 and AZD6244 (Selumetinib) are drugs currently under clinical investigation for cancer treatment, however the Ras–MAPK pathway is also an important mediator of normal bone cell differentiation and function. In this study we examined the effects of these compounds on endochondral processes using both in vitro and in vivo models. Treatment with PD0325901 or AZD6244 significantly increased Runx2 and Alkaline phosphate gene expression in calvarial osteoblasts and decreased TRAP + cells in induced osteoclast cultures. To test the effects of these drugs on bone healing, C57/Bl6 mice underwent a closed tibial fracture and were treated with PD0325901 or AZD6244 at 10 mg/kg/day. Animals were culled at day 10 and at day 21 post-fracture for analysis of the fracture callus and the femoral growth plate in the contralateral leg. MEKi treatment markedly increased cartilage volume in the soft callus at day 10 post-fracture (+ 60% PD0325901, + 20% AZD6244) and continued treatment led to a delay in cartilage remodeling. At the growth plate, we observed an increase in the height of the hypertrophic zone relative to the proliferative zone of + 78% in PD0325901 treated mice. Osteoclast surface was significantly decreased both at the terminal end of the growth plate and within the fracture calluses of MEKi treated animals. The mechanistic effects of MEKi on genes encoding cartilage matrix proteins and catabolic enzymes were examined in articular chondrocyte cultures. PD0325901 or AZD6244 led to increased matrix protein expression (Col2a1 and Acan) and decreased expression of catabolic factors (Mmp13 and Adamts-5). Taken together, these data support the hypothesis that MEKi treatment can impact chondrocyte hypertrophy, matrix resorption, and fracture healing. These compounds can also affect bone architecture by expanding the hypertrophic zone of the growth plate and reducing osteoclast surface systemically.     

Low osteocalcin/collagen type I bone gene expression ratio is associated with hip fragility fractures

IntroductionOsteocalcin (OC) is the most abundant non-collagenous bone protein and is determinant for bone mineralization.We aimed to compare OC bone expression and serum factors related to its carboxylation in hip fragility fracture and osteoarthritis patients. We also aimed to identify which of these factors were associated with worse mechanical behavior and with the hip fracture event.MethodsIn this case-control study, fragility fracture patients submitted to hip replacement surgery were evaluated and compared to a group of osteoarthritis patients submitted to the same procedure. Fasting blood samples were collected to assess apolipoproteinE (apoE) levels, total OC and undercarboxylated osteocalcin (ucOC), vitamin K, LDL cholesterol, triglycerides and bone turnover markers. The frequency of the apoε4 isoform was determined.Femoral epiphyses were collected and trabecular bone cylinders drilled in order to perform compression mechanical tests. Gene expression of bone matrix components was assessed by quantitative RT-PCR analysis.Results64 patients, 25 submitted to hip replacement surgery due to fragility fracture and 39 due to osteoarthritis, were evaluated. Bone OC/collagen expression (OC/COL1A1) ratio was significantly lower in hip fracture compared to osteoarthritis patients (p < 0.017) adjusted for age, gender and body mass index. Moreover, OC/COL1A1 expression ratio was associated with the hip fracture event (OR ~ 0; p = 0.003) independently of the group assigned, or the clinical characteristics. Apoε4 isoform was more frequent in the hip fracture group (p = 0.029). ucOC levels were higher in the fracture group although not significantly (p = 0.058). No differences were found regarding total OC (p = 0.602), apoE (p = 0.467) and Vitamin K (p = 0.371).In hip fracture patients, multivariate analysis, adjusted for clinical characteristics, serum factors related to OC metabolism and gene expression of bone matrix proteins showed that low OC/COL1A1 expression ratio was significantly associated with worse trabecular strength (β = 0.607; p = 0.013) and stiffness (β = 0.693; p = 0.003). No association was found between ucOC and bone mechanics. Moreover, in osteoarthritis patients, the multivariate analysis revealed that serum total OC was negatively associated with strength (β = ? 0.411; p = 0.030) and stiffness (β = ? 0.487; p = 0.009).ConclusionWe demonstrated that low bone OC/COL1A1 expression ratio was an independent predictor of worse trabecular mechanical behavior and of the hip fracture event. These findings suggest that in hip fracture patients the imbalance of bone OC/COL1A1 expression ratio reflects disturbances in osteoblast activity leading to bone fragility.