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.     

Conditional deletion of IGF-I in osteocytes unexpectedly accelerates bony union of the fracture gap in mice

This study evaluated the effects of deficient IGF-I expression in osteocytes on fracture healing. Transgenic mice with conditional knockout (cKO) of Igf1 in osteocytes were generated by crossing Dmp1-Cre mice with Igf1 flox mice. Fractures were created on the mid-shaft of tibia of 12-week-old male cKO mice and wild-type (WT) littermates by three-point bending. At 21 and 28 days post-fracture healing, the increases in cortical bone mineral density, mineral content, bone area, and thickness, as well as sub-cortical bone mineral content at the fracture site were each greater in cKO calluses than in WT calluses. There were 85% decrease in the cartilage area and > 2-fold increase in the number of osteoclasts in cKO calluses at 14 days post-fracture, suggesting a more rapid remodeling of endochondral bone. The upregulation of mRNA levels of osteoblast marker genes (cbfa1, alp, Opn, and Ocn) was greater in cKO calluses than in WT calluses. μ-CT analysis suggested an accelerated bony union of the fracture gap in cKO mice. The Sost mRNA level was reduced by 50% and the Bmp2 mRNA level was increased 3-fold in cKO fractures at 14 days post-fracture, but the levels of these two mRNAs in WT fractures were unchanged, suggesting that the accelerated fracture repair may in part act through the Wnt and/or BMP signaling. In conclusion, conditional deletion of Igf1 in osteocytes not only did not impair, but unexpectedly enhanced, bony union of the fracture gap. The accelerated bony union was due in part to upregulation of the Wnt and BMP2 signaling in response to deficient osteocyte-derived IGF-I expression, which in turn favors intramembranous over endochondral bone repair.     

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.     

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.     

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.     

Assessment of the healing process in distal radius fractures by high resolution peripheral quantitative computed tomography

In clinical practice, fracture healing is evaluated by clinical judgment in combination with conventional radiography. Due to limited resolution, radiographs don't provide detailed information regarding the bone micro-architecture and bone strength. Recently, assessment of in vivo bone density, architectural and mechanical properties at the microscale became possible using high resolution peripheral quantitative computed tomography (HR-pQCT) in combination with micro finite element analysis (μFEA). So far, such techniques have been used mainly to study intact bone. The aim of this study was to explore whether these techniques can also be used to assess changes in bone density, micro-architecture and bone stiffness during fracture healing. Therefore, the fracture region in eighteen women, aged 50 years or older with a stable distal radius fracture, was scanned using HR-pQCT at 1–2 (baseline), 3–4, 6–8 and 12 weeks post-fracture. At 1–2 and 12 weeks post-fracture the distal radius at the contra-lateral side was also scanned as control. Standard bone density, micro-architectural and geometric parameters were calculated and bone stiffness in compression, torsion and bending was assessed using μFEA. A linear mixed effect model with time post-fracture as fixed effect was used to detect significant (p-value ≤ 0.05) changes from baseline. Wrist pain and function were scored using the patient-rated wrist evaluation (PRWE) questionnaire. Correlations between the bone parameters and the PRWE score were calculated by Spearman's correlation coefficient. At the fracture site, total and trabecular bone density increased by 11% and 20%, respectively, at 6–8 weeks, whereas cortical density was decreased by 4%. Trabecular thickness increased by 23–31% at 6–8 and 12 weeks and the intertrabecular area became blurred, indicating intertrabecular bone formation. Compared to baseline, calculated bone stiffness in compression, torsion and bending was increased by 31% after 12 weeks. A moderate negative correlation was found between the stiffness and the PRWE score. No changes were observed at the contra-lateral side. The results demonstrate that it is feasible to assess clinically relevant and significant longitudinal changes in bone density, micro-architecture and mechanical properties at the fracture region during the healing process of stable distal radius fractures using HR-pQCT.     

Sex-specific compromised bone healing in female rats might be associated with a decrease in mesenchymal stem cell quantity

IntroductionThe clinically known importance of patient sex as a major risk factor for compromised bone healing is poorly reflected in animal models. Consequently, the underlying cellular mechanisms remain elusive. Because mesenchymal stem cells (MSCs) are postulated to regulate tissue regeneration and give rise to essential differentiated cell types, they may contribute to sex-specific differences in bone healing outcomes.MethodsWe investigated sex-specific variations in bone healing and associated differences in MSC populations. A 1.5 mm osteotomy gap in the femora of 8 male and 8 female 12-month-old Sprague–Dawley rats was stabilized by an external fixator. Healing was analyzed in terms of biomechanical testing, bridging and callus size over time (radiography at 2, 4, and 6 weeks after surgery), and callus volume and geometry by μCT at final follow-up. MSCs were obtained from bone marrow samples of an age-matched group of 12 animals (6 per gender) and analyzed for numbers of colony-forming units (CFUs) and their capacity to differentiate and proliferate. The proportion of senescent cells was determined by β-galactosidase staining.ResultsSex-specific differences were indicated by a compromised mechanical competence of the callus in females compared with males (maximum torque at failure, p = 0.028). Throughout the follow-up, the cross-sectional area of callus relative to bone was reduced in females (p ≤ 0.01), and the bridging of callus was delayed (p_2weeks = 0.041). μCT revealed a reduced callus size (p = 0.003), mineralization (p = 0.003) and polar moment of inertia (p = 0.003) in female animals. The female bone marrow contained significantly fewer MSCs, represented by low CFU numbers in both femora and tibiae (p_femur = 0.017, p_tibia = 0.010). Functional characteristics of male and female MSCs were similar.ConclusionBiomechanically compromised and radiographically delayed bone formation were distinctive in female rats. These differences were concomitant with a reduced number of MSCs, which may be causative for the suboptimal bone healing.     

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.     

Teriparatide improves bone quality and healing of atypical femoral fractures associated with bisphosphonate therapy

Bone remodelling suppressants like the bisphosphonates reduce bone loss and slow progression of structural decay. As remodelling removes damaged bone, when remodelling suppression is protracted, bone quality may be compromised predisposing to microdamage accumulation and atypical femoral fractures. The aim of this study was to determine whether teriparatide therapy assists in fracture healing and improves bone quality in patients with bisphosphonate associated atypical femoral fractures.A prospective study was conducted involving 14 consecutive patients presenting during 2 years with atypical femoral fracture. All patients were offered teriparatide therapy unless contraindicated. Age and sex matched control subjects without fragility fractures or anti-resorptive treatment were recruited. High resolution peripheral micro-computed tomography (HRpQCT) scans of the distal radius and distal tibia were analysed for their cortical bone tissue mineralisation density using new software (StrAx1.0, StrAxCorp, Australia) at baseline and 6 months after teriparatide.Administration of 20 μg of teriparatide subcutaneously daily for 6 months to 5 of the 14 patients was associated with 2–3 fold increase in bone remodelling markers (p = 0.01) and fracture healing. At the distal radius, the proportion of less densely mineralised bone increased by 29.5% (p = 0.01), and the proportion of older, more densely mineralised bone decreased by 16.2% (p = 0.03). Similar observations were made at the distal tibia. Of the nine patients managed conservatively or surgically, seven had poor fracture healing with ongoing pain, one sustained a contralateral atypical fracture and one had fracture union after 1 year. Teriparatide may assist in healing of atypical fractures and restoration of bone quality.     

Influences of age and mechanical stability on volume,microstructure, and mineralization of the fracture callus during bone healing: Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague–Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger ΔCSA (Callus cross-sectional area) compared to rigid groups. In vitro µCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (p < 0.01). Younger animals showed (i) larger callus strut thickness (p < 0.001), (ii) lower perforation in struts (p < 0.01), and (iii) higher mineralization of callus struts (p < 0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (p < 0.05). Blood vessel density was reduced in animals with semirigid fixation (p < 0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.     

Knee loading protects against osteonecrosis of the femoral head by enhancing vessel remodeling and bone healing

Osteonecrosis of the femoral head is a serious orthopedic problem. Moderate loads with knee loading promote bone formation, but their effects on osteonecrosis have not been investigated. Using a rat model, we examined a hypothesis that knee loading enhances vessel remodeling and bone healing through the modulation of the fate of bone marrow-derived cells. In this study, osteonecrosis was induced by transecting the ligamentum teres followed by a tight ligature around the femoral neck. For knee loading, 5 N loads were laterally applied to the knee at 15 Hz for 5 min/day for 5 weeks. Changes in bone mineral density (BMD) and bone mineral content (BMC) of the femur were measured by pDEXA, and ink infusion was performed to evaluate vessel remodeling. Femoral heads were harvested for histomorphometry, and bone marrow-derived cells were isolated to examine osteoclast development and osteoblast differentiation. The results showed that osteonecrosis significantly induced bone loss, and knee loading stimulated both vessel remodeling and bone healing. The osteonecrosis group exhibited the lowest trabecular BV/TV (p < 0.001) in the femoral head, and lowest femoral BMD and BMC (both p < 0.01). However, knee loading increased trabecular BV/TV (p < 0.05) as well as BMD (p < 0.05) and BMC (p < 0.01). Osteonecrosis decreased the vessel volume (p < 0.001), vessel number (p < 0.001) and VEGF expression (p < 0.01), and knee loading increased them (p < 0.001, p < 0.001 and p < 0.01). Osteonecrosis activated osteoclast development, and knee loading reduced its formation, migration, adhesion and the level of “pit” formation (p < 0.001, p < 0.01, p < 0.001 and p < 0.001). Furthermore, knee loading significantly increased osteoblast differentiation and CFU-F (both p < 0.001). A significantly positive correlation was observed between vessel remodeling and bone healing (both p < 0.01). These results indicate that knee loading could be effective in repair osteonecrosis of the femoral head in a rat model. This effect might be attributed to promoting vessel remodeling, suppressing osteoclast development, and increasing osteoblast and fibroblast differentiation. In summary, the current study suggests that knee loading might potentially be employed as a non-invasive therapy for osteonecrosis of the femoral head.     

Hedgehog signaling mediates woven bone formation and vascularization during stress fracture healing

Hedgehog (Hh) signaling is critical in developmental osteogenesis, and recent studies suggest it may also play a role in regulating osteogenic gene expression in the post-natal setting. However, there is a void of studies directly assessing the effect of Hh inhibition on post-natal osteogenesis. This study utilized a cyclic loading-induced ulnar stress fracture model to evaluate the hypothesis that Hh signaling contributes to osteogenesis and angiogenesis during stress fracture healing. Immediately prior to loading, adult rats were given GDC-0449 (Vismodegib — a selective Hh pathway inhibitor; 50 mg/kg orally twice daily), or vehicle. Hh signaling was upregulated in response to stress fracture at 3 days (Ptch1, Gli1 expression), and was markedly inhibited by GDC-0449 at 1 day and 3 days in the loaded and non-loaded ulnae. GDC-0449 did not affect Hh ligand expression (Shh, Ihh, Dhh) at 1 day, but decreased Shh expression by 37% at 3 days. GDC-0449 decreased woven bone volume (− 37%) and mineral density (− 17%) at 7 days. Dynamic histomorphometry revealed that the 7 day callus was composed predominantly of woven bone in both groups. The observed reduction in woven bone occurred concomitantly with decreased expression of Alpl and Ibsp, but was not associated with differences in early cellular proliferation (as determined by callus PCNA staining at 3 days), osteoblastic differentiation (Osx expression at 1 day and 3 days), chondrogenic gene expression (Acan, Sox9, and Col2α1 expression at 1 day and 3 days), or bone resorption metrics (callus TRAP staining at 3 days, Rankl and Opg expression at 1 day and 3 days). To evaluate angiogenesis, vWF immunohistochemistry showed that GDC-0449 reduced fracture callus blood vessel density by 55% at 3 days, which was associated with increased Hif1α gene expression (+ 30%). Dynamic histomorphometric analysis demonstrated that GDC-0449 also inhibited lamellar bone formation. Lamellar bone analysis of the loaded limb (directly adjacent to the woven bone callus) showed that GDC-0449 significantly decreased mineral apposition rate (MAR) and bone formation rate (BFR/BS) (− 17% and − 20%, respectively). Lamellar BFR/BS in the non-loaded ulna was also significantly decreased (− 37%), indicating that Hh signaling was required for normal bone modeling. In conclusion, Hh signaling plays an important role in post-natal osteogenesis in the setting of stress fracture healing, mediating its effects directly through regulation of bone formation and angiogenesis.     

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.     

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.     

Multiscale alterations in bone matrix quality increased fragility in steroid induced osteoporosis

A serious adverse clinical effect of glucocorticoid steroid treatment is secondary osteoporosis, enhancing fracture risk in bone. This rapid increase in bone fracture risk is largely independent of bone loss (quantity), and must therefore arise from degradation of the quality of the bone matrix at the micro- and nanoscale. However, we lack an understanding of both the specific alterations in bone quality n steroid-induced osteoporosis as well as the mechanistic effects of these changes. Here we demonstrate alterations in the nanostructural parameters of the mineralized fibrillar collagen matrix, which affect bone quality, and develop a model linking these to increased fracture risk in glucocorticoid induced osteoporosis. Using a mouse model with an N-ethyl-N-nitrosourea (ENU)-induced corticotrophin releasing hormone promoter mutation (Crh^− 120/+) that developed hypercorticosteronaemia and osteoporosis, we utilized in situ mechanical testing with small angle X-ray diffraction, synchrotron micro-computed tomography and quantitative backscattered electron imaging to link altered nano- and microscale deformation mechanisms in the bone matrix to abnormal macroscopic mechanics. We measure the deformation of the mineralized collagen fibrils, and the nano-mechanical parameters including effective fibril modulus and fibril to tissue strain ratio. A significant reduction (51%) of fibril modulus was found in Crh^− 120/+ mice. We also find a much larger fibril strain/tissue strain ratio in Crh^− 120/+ mice (~ 1.5) compared to the wild-type mice (~ 0.5), indicative of a lowered mechanical competence at the nanoscale. Synchrotron microCT show a disruption of intracortical architecture, possibly linked to osteocytic osteolysis. These findings provide a clear quantitative demonstration of how bone quality changes increase macroscopic fragility in secondary osteoporosis.     

Evidence for reduced bone formation surface relative to bone resorption surface in female femoral fragility fracture patients

Fragility fractures, including neck of femur fractures, result from reductions in the amount, quality and architecture of bone. The aim of this study was to compare the cancellous bone structure, and static indices of bone turnover, in female patients, who had sustained fragility fracture at the femoral neck, with age-matched females without fragility fracture. Bone samples were taken from the intertrochanteric region of the proximal femur of female patients undergoing hip arthroplasty surgery for a subcapital fragility fracture of the femoral neck (#NOF) or from age-matched female control individuals at routine autopsy. Contiguous bone samples were analyzed for undecalcified histomorphometry and for mRNA expression. The histomorphometric data, which were normally distributed, indicated no difference between the mean values for any of the structural parameters in control and fracture samples. In particular, the bone volume (BV/TV) values were not different and did not change significantly with age in these cohorts of individuals aged > 65 years. The static indices of bone turnover, eroded surface (ES/BS) and osteoid surface (OS/BS), were positively correlated with age in the > 65-year-old control group (p < 0.055 and p < 0.03, respectively). The median values for these indices were not different between the fracture and control groups. However, both the median and the range of OS/BS values were increased for > 65-year-old controls compared with a group of younger females aged < 65 years, suggesting an increase in bone formation surface in older females in the proximal femur after 65 years of age. When the data were further interrogated, a reduction in the percentage osteoid surface to eroded surface quotient (OS/ES) was found for the fracture group compared with the age-matched control group suggesting a reduced adaptive modeling drift capability in the fracture group. In contiguous bone samples, increased median values for receptor activator of nuclear factor kappa β (RANK) and interleukin-6 (IL-6) mRNA expression were observed in the fracture group. Study of cultured human osteoblasts showed that recombinant human IL-6 (rhIL-6) inhibited osteoblast differentiation, as measured by an increase in the immature osteoblast marker, STRO-1 and concomitantly decreased expression of the osteoblast maturation marker, alkaline phosphatase. Importantly, cells cultured in the presence of IL-6 showed significantly less mineral deposition in vitro compared with control cultures. These data suggest that perturbations in bone formation surface, relative to resorption surface, are potentially important in producing bone in the proximal femur with increased propensity to fracture.     

Localized deferoxamine injection augments vascularity and improves bony union in pathologic fracture healing after radiotherapy

BackgroundMedically based efforts and alternative treatment strategies to prevent or remediate the corrosive effects of radiotherapy on pathologic fracture healing have failed to produce clear and convincing evidence of success. Establishing an effective pharmacologic option to prevent or treat the development of non-unions in this setting could have immense therapeutic potential. Experimental studies have shown that deferoxamine (DFO), an iron-chelating agent, bolsters vascularity and subsequently enhances normal fracture healing when injected locally into a fracture callus in long bone animal models. Since radiotherapy is known to impede angiogenesis, we hypothesized that the pharmacologic addition of DFO would serve to mitigate the effects of radiotherapy on new vessel formation in vitro and in vivo.Materials and MethodsIn vitro investigation of angiogenesis was conducted utilizing HUVEC cells in Matrigel. Endothelial tubule formation assays were divided into four groups: Control, Radiated, Radiated + Low-Dose DFO and Radiated + High-Dose DFO. Tubule formation was quantified microscopically and video recorded for the four groups simultaneously during the experiment. In vivo, three groups of Sprague–Dawley rats underwent external fixator placement and fracture osteotomy of the left mandible. Two groups received pre-operative fractionated radiotherapy, and one of these groups was treated with DFO after fracture repair. After 40 days, the animals were perfused and imaged with micro-CT to calculate vascular radiomorphometrics.ResultsIn vitro, endothelial tubule formation assays demonstrated that DFO mitigated the deleterious effects of radiation on angiogenesis. Further, high-dose DFO cultures appeared to organize within 2 h of incubation and achieved a robust network that was visibly superior to all other experimental groups in an accelerated fashion. In vivo, animals subjected to a human equivalent dose of radiotherapy (HEDR) and left mandibular fracture demonstrated quantifiably diminished μCT metrics of vascular density, as well as a 75% incidence of associated non-unions. The addition of DFO in this setting markedly improved vascularity as demonstrated with 3D angiographic modeling. In addition, we observed an increased incidence of bony unions in the DFO treated group when compared to radiated fractures without treatment (67% vs. 25% respectively).ConclusionOur data suggest that selectively targeting angiogenesis with localized DFO injections is sufficient to remediate the associated severe vascular diminution resulting from a HEDR. Perhaps the most consequential and clinically relevant finding was the ability to reduce the incidence of non-unions in a model where fracture healing was not routinely observed.     

Bone Mineral Density in Premenopausal Arab Women With Type 2 Diabetes Mellitus

IntroductionThis study compares an ethnically uniform group of premenopausal type 2 diabetic (T2DM) Arab women with a matched control group of nondiabetic subjects, in terms of their bone mineral density (BMD) and anthropometric measurements.MethodsThe study included 252 premenopausal Arab women. Their age ranged from 26 to 50 yr with a mean ± SD of 43.65 ± 8.97 yr. One hundred and twenty-two women were T2DM patients and 130 women were nondiabetic controls. The controls matched the subjects in gender, age, and body mass index (BMI). BMD was measured at total lumbar spine (L1–L4) and total left hip, using dual-energy X-ray absorptiometry (DXA; HOLOGIC, QRS SERIES, Europe, Belgium). Difference in BMD and its relationship to the anthropometric measurements in T2DM and control groups were assessed.ResultsSignificant difference was found between T2DM patients and nondiabetic patients in their mean hip BMD (0.92 ± 0.16 vs. 0.87 ± 0.14, p < 0.05) and spine BMD (0.93 ± 0.15 vs. 0.88 ± 0.14, p < 0.01). No significant difference was found in age, height, weight, and BMI (p > 0.05). The increase in hip BMD in T2DM patients normalized and the increase in spine BMD persisted after controlling for the confounding effect of age and anthropometric measurements.ConclusionPremenopausal Arab women with T2DM have higher BMD at the spine than women without T2DM. The underlying mechanism causing this increase does not seem to be related to ethnicity, gender, hormonal status, or anthropometric measurements.     

Effect of combined treatment with zoledronic acid and parathyroid hormone on mouse bone callus structure and composition

In recent years, great interest in combined treatment of parathyroid hormone (PTH) with anti-resorptive therapy has emerged. PTH has been suggested to aid bridging of atrophic fractures and improve strength in closed fracture models. Bisphosphonate treatments typically result in a larger woven bone callus that is slower to remodel. The combination of both drugs has been demonstrated to be effective for the treatment of osteoporotic bone loss in many preclinical studies. However, the effect of combined treatment on fracture repair is still largely unexplored. In this study, we aimed to compare these drugs as single-agent and in combination in a murine closed fracture model. We wanted to assess potential differences in material properties, morphometry and in the development of the lacuno-canalicular network. A total of 40 female, 11-week-old wild type mice underwent a closed fracture on the midshaft of the tibia and were assigned to four groups (n = 8–10 per group). Beginning on post-operative day 8, animals received different subcutaneous injections. Group 1 received a single injection of saline solution and Group 2 of zoledronic acid (ZA). Group 3 received daily dosing of PTH. Group 4 received a dual treatment, starting with a single dose of ZA followed by daily injection of PTH. Three weeks after fracture, all animals were euthanized and tibiae were assessed using micro-computed tomography (micro-CT), high-resolution micro-CT (HR micro-CT), Raman spectroscopy, quantitative histomorphometry, and deconvolution microscopy (DV microscopy). Combined treatment showed a significant increase of 41% in bone volume fraction and a significant decrease of 61% in the standard deviation of the trabecular spacing compared to vehicle, both known to be strong predictors of callus strength. An analysis via HR micro-CT showed similar results on all groups for lacunar numerical density, whereas mean lacuna volume was found to be higher compared to vehicle in treated groups, but only PTH mono-treatment showed a significant increase compared to vehicle (+ 45%). Raman spectroscopy did not reveal detectable changes in material properties of the bone calluses. Sclerostin staining, tartrate resistant acid phosphatase (TRAP) staining and canalicular analysis with DV microscopy on a subset of samples did not display distinctive difference in any of the treatments.We therefore consider PTH + ZA treatment beneficial for bone healing. No clear negative effect on bone quality was detected during this study.