Loss of milk fat globule-epidermal growth factor 8 (MFG-E8) in mice leads to low bone mass and accelerates ovariectomy-associated bone loss by increasing osteoclastogenesis

Milk fat globule-epidermal growth factor 8 (MFG-E8) is a glycoprotein that controls the engulfment of apoptotic cells and exerts inflammation-modulatory effects. Recently, it has been implicated in osteoclastogenesis and the pathogenesis of inflammatory periodontal bone loss, but its role in physiological bone homeostasis is still not well defined. Here, we evaluated the influence of MFG-E8 on osteoblasts and osteoclasts and its impact on bone remodeling in healthy and ovariectomized mice as a model for post-menopausal osteoporosis.Total and trabecular bone mineral densities at the lumbar spine in 6-week-old MFG-E8 KO mice were reduced by 11% (p < 0.05) and 17% (p < 0.01), respectively, as compared to wild-type (WT) mice. Accordingly, serum levels of the bone formation marker P1NP were decreased by 37% (p < 0.01) in MFG-E8 KO mice as were the ex vivo mineralization capacity and expression of osteoblast genes (Runx2, alkaline phosphatase, osteocalcin) in MFG-E8 KO osteoblasts. In contrast, serum bone resorption markers CTX1 and TRAP5b were increased by 30% and 60% (p < 0.05), respectively, in MFG-E8 KO mice. Furthermore, bone marrow macrophages from MFG-E8-KO mice differentiated more effectively into osteoclasts, as compared to WT cells. MFG-E8-deficient osteoclasts displayed increased bone resorption ex vivo, which could be reversed by the presence of recombinant MFG-E8. To determine the significance of the enhanced osteoclastogenesis in MFG-E8 KO mice, we performed an ovariectomy, which is associated with bone loss due to increased osteoclast activity. Indeed, MFG-E8 KO mice lost 12% more trabecular bone density than WT mice after ovariectomy.Together, these data indicate that MFG-E8 controls steady-state and pathological bone turnover and may therefore represent a new target gene in the treatment of bone diseases.     

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.     

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.     

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.     

Effects of parathyroid hormone on cortical porosity,non-enzymatic glycation and bone tissue mechanics in rats with type 2 diabetes mellitus

Type 2 diabetes mellitus increases skeletal fragility; however, the contributing mechanisms and the efficacy of bone-forming agents are unclear. We studied diabetes and parathyroid hormone (PTH) treatment effects on cortical porosity (Ct.Po), non-enzymatic glycation (NEG) and bone mechanics in Zucker diabetic fatty (ZDF) rats.Eleven-week old ZDF diabetic (DB) and non-diabetic (ND) rats were given 75 μg/kg PTH (1–84) or vehicle 5 days per week over 12 weeks. The right femora and L4 vertebrae were excised, micro-CT scanned, and tested in 3-point bending and uniaxial compression, respectively. NEG of the samples was determined using fluorescence.Diabetes increased Ct.Po (vertebra (vert): + 40.6%, femur (fem): + 15.5% vs. ND group, p < 0.05) but had no effect on NEG. PTH therapy reduced vertebral NEG in the ND animals only (− 73% vs untreated group, p < 0.05), and increased femoral NEG in the DB vs. ND groups (+ 63%, p < 0.05). PTH therapy had no effect on Ct.Po. Diabetes negatively affected bone tissue mechanics where reductions in vertebral maximum strain (− 22%) and toughness (− 42%) were observed in the DB vs. ND group (p < 0.05). PTH improved maximum strain in the vertebra of the ND animals (+ 21%, p < 0.05) but did not have an effect in the DB group. PTH increased femoral maximum strain (+ 21%) and toughness (+ 28%) in ND and decreased femoral maximum stress (− 13%) and toughness (− 27%) in the DB animals (treated vs. untreated, p < 0.05). Ct.Po correlated negatively with maximum stress (fem: R = − 0.35, p < 0.05, vert: R = − 0.57, p < 0.01), maximum strain (fem: R = − 0.35, p < 0.05, vert: R = − 0.43, p < 0.05) and toughness (fem: R = − 0.34, p < 0.05, vert: R = − 0.55, p < 0.01), and NEG correlated negatively with toughness at the femur (R = − 0.34, p < 0.05) and maximum strain at the vertebra (R = − 0.49, p < 0.05).Diabetes increased cortical porosity and reduced bone mechanics, which were not improved with PTH treatment. PTH therapy alone may worsen diabetic bone mechanics through formation of new bone with high AGEs cross-linking. Optimal treatment regimens must address both improvements of bone mass and glycemic control in order to successfully reduce diabetic bone fragility.This article is part of a Special Issue entitled “Bone and diabetes”.     

Fractures on bisphosphonates in osteoporosis pseudoglioma syndrome (OPPG): pQCT shows poor bone density and structure

Osteoporosis pseudoglioma syndrome (OPPG) is a rare autosomal recessive disorder of childhood osteoporosis and blindness due to inactivating mutations in LDL receptor-like protein 5 (LRP5). We and others have reported improvement in areal bone mineral density (aBMD) by DXA in OPPG on short term bisphosphonates. Long-term data on bisphosphonate use in OPPG and measures of volumetric BMD (vBMD) and cortical structure are not available. In addition, no long-term DXA data on untreated OPPG is available. The aims of this study were to: (1) record low trauma fractures and longitudinal aBMD by DXA in 5 OPPG patients on chronic bisphosphonate treatment, and in 4 OPPG patients never treated (2) to perform tibia peripheral quantitative CT (pQCT) to evaluate volumetric bone mineral density (vBMD), cortical structure and calf muscle area in 6 OPPG patients and 14 unaffected first degree family members. pQCT results were converted to sex-specific Z-scores for age and adjusted for tibia length based on data in > 700 reference participants. We observed 4 fractures (3 femoral shafts) in 3 OPPG patients while on bisphosphonates, after each achieved significant improvement in aBMD. OPPG participants had significantly lower mean trabecular vBMD (− 1.51 vs. 0.17, p = 0.002), cortical area (− 2.36 vs. 0.37; p < 0.001) and periosteal circumference (− 1.86 vs. − 0.31, p = 0.001) Z-scores, compared with unaffected participants and had a trend toward lower muscle area Z-score (− 0.69 vs. 0.47, p = 0.12). These data demonstrate substantial bone fragility despite improvements in aBMD. The pQCT data provide insight into the fragility with substantial deficits in trabecular vBMD and cortical dimensions, consistent with OPPG effects of bone formation. Treatment that improves bone quality is needed to reduce fractures in OPPG.     

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.     

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.     

Glucose-dependent insulinotropic polypeptide (GIP) receptor deletion leads to reduced bone strength and quality

Bone is permanently remodeled by a complex network of local, hormonal and neuronal factors that affect osteoclast and osteoblast biology. In this context, a role for gastro-intestinal hormones has been proposed based on evidence that bone resorption dramatically falls after a meal. Glucose-dependent insulinotropic polypeptide (GIP) is one of the candidate hormones as its receptor, glucose-dependent insulinotropic polypeptide receptor (GIPR), is expressed in bone. In the present study we investigated bone strength and quality by three-point bending, quantitative x-ray microradiography, microCT, qBEI and FTIR in a GIPR knockout (GIPR KO) mouse model and compared with control wild-type (WT) animals. Animals with a deletion of the GIPR presented with a significant reduction in ultimate load (-− 11%), stiffness (− 16%), total absorbed (− 28%) and post-yield energies (− 27%) as compared with WT animals. Furthermore, despite no change in bone outer diameter, the bone marrow diameter was significantly increased and as a result cortical thickness was significantly decreased by 20% in GIPR deficient animals. Bone resorption at the endosteal surface was significantly increased whilst bone formation was unchanged in GIPR deficient animals. Deficient animals also presented with a pronounced reduction in the degree of mineralization of bone matrix. Furthermore, the amount of mature cross-links of collagen matrix was significantly reduced in GIPR deficient animals and was associated with lowered intrinsic material properties. Taken together, these data support a positive effect of the GIPR on bone strength and quality.     

The resistance of cortical bone tissue to failure under cyclic loading is reduced with alendronate

Bisphosphonates are the most prescribed preventative treatment for osteoporosis. However, their long-term use has recently been associated with atypical fractures of cortical bone in patients who present with low-energy induced breaks of unclear pathophysiology. The effects of bisphosphonates on the mechanical properties of cortical bone have been exclusively studied under simple, monotonic, quasi-static loading. This study examined the cyclic fatigue properties of bisphosphonate-treated cortical bone at a level in which tissue damage initiates and is accumulated prior to frank fracture in low-energy situations. Physiologically relevant, dynamic, 4-point bending applied to beams (1.5 mm × 0.5 mm × 10 mm) machined from dog rib (n = 12/group) demonstrated mechanical failure and micro-architectural features that were dependent on drug dose (3 groups: 0, 0.2, 1.0 mg/kg/day; alendronate [ALN] for 3 years) with cortical bone tissue elastic modulus (initial cycles of loading) reduced by 21% (p < 0.001) and fatigue life (number of cycles to failure) reduced in a stress-life approach by greater than 3-fold with ALN1.0 (p < 0.05). While not affecting the number of osteons, ALN treatment reduced other features associated with bone remodeling, such as the size of osteons (− 14%; ALN1.0: 10.5 ± 1.8, VEH: 12.2 ± 1.6, × 10³ μm²; p < 0.01) and the density of osteocyte lacunae (− 20%; ALN1.0: 11.4 ± 3.3, VEH: 14.3 ± 3.6, × 10² #/mm²; p < 0.05). Furthermore, the osteocyte lacunar density was directly proportional to initial elastic modulus when the groups were pooled (R = 0.54, p < 0.01). These findings suggest that the structural components normally contributing to healthy cortical bone tissue are altered by high-dose ALN treatment and contribute to reduced mechanical properties under cyclic loading conditions.     

Risedronate may preserve bone microarchitecture in breast cancer survivors on aromatase inhibitors: A randomized,controlled clinical trial

This study provides preliminary evidence that risedronate not only preserves BMD but may also attenuate the loss of bone microarchitecture over 2 years during a time of accelerated bone loss in post-menopausal breast cancer survivors on aromatase inhibitors.IntroductionAccelerated bone loss and elevated fracture risk are associated with the use of aromatase inhibitors (AIs) in women with breast cancer. We previously reported that the oral bisphosphonate, risedronate, can maintain bone mineral density (BMD) in the hip and spine over 2-years in post-menopausal breast cancer survivors on AIs. In this study, we examined whether oral bisphosphonates can also preserve bone microarchitecture as measured by the trabecular bone score (TBS) in this population.MethodsThis 2-year randomized, double-blind, placebo-controlled trial included postmenopausal women over age 55 with breast cancer on an AI who had low bone mass. Participants provided informed consent and were randomized to risedronate 35 mg once weekly or placebo. We examined 12- and 24-month changes in spine TBS, analyzed using linear mixed models.ResultsOne-hundred and nine women with a mean age of 70.5 years were included in the analysis. In the placebo group, BMD declined at the spine and hip over the 24-month period but was preserved in the active treatment group (data previously reported). TBS declined in the placebo group by − 2.1% and − 2.3% at 12- and 24-months, respectively (p < 0.005). The TBS percent change in bisphosphonate-treated patients was − 0.9% and − 1.3% at 12 and 24-months but did not reach statistical significance (p = 0.24 and 0.14). The 12- and 24-month between-group differences were 0.9 (p = 0.38) and 0.8 (p = 0.44) percentage points. TBS change correlated with spine BMD changes in the placebo group at 12- and 24-months (r = 0.33 and 0.34, p < 0.01) but not in the active treatment group.ConclusionThe oral bisphosphonate risedronate preserves BMD and may attenuate loss of bone microarchitecture over 2 years during a time of accelerated bone loss in breast cancer survivors on AIs, but more definitive evidence is needed.     

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.     

Embryonic stem cell therapy improves bone quality in a model of impaired fracture healing in the mouse; tracked temporally using in vivo micro-CT

In the current study, we used an estrogen-deficient mouse model of osteoporosis to test the efficacy of a cell-generated bone tissue construct for bone augmentation of an impaired healing fracture. A reduction in new bone formation at the defect site was observed in ovariectomized fractures compared to the control group using repeated measures in vivo micro-computed tomography (μCT) imaging over 4 weeks. A significant increase in the bone mineral density (BMD), trabecular bone volume ratio, and trabecular number, thickness and connectivity were associated with fracture repair in the control group, whereas the fractured bones of the ovariectomized mice exhibited a loss in all of these parameters (p < 0.001). In a separate group, ovariectomized fractures were treated with murine embryonic stem (ES) cell-derived osteoblasts loaded in a three-dimensional collagen I gel and recovery of the bone at the defect site was observed. A significant increase in the trabecular bone volume ratio (p < 0.001) and trabecular number (p < 0.01) was observed by 4 weeks in the fractures treated with cell-loaded collagen matrix compared to those treated with collagen I alone. The stem cell-derived osteoblasts were identified at the fracture site at 4 weeks post-implantation through in situ hybridization histochemistry. Although this cell tracking method was effective, the formation of an ectopic cellular nodule adjacent to the knee joints of two mice suggested that alternative in vivo cell tracking methods should be employed in order to definitively assess migration of the implanted cells. To our knowledge, this study is the first of its kind to examine the efficacy of stem cell therapy for fracture repair in an osteoporosis-related fracture model in vivo. The findings presented provide novel insight into the use of stem cell therapies for bone injuries.     

Bone's responses to mechanical loading are impaired in type 1 diabetes

Diabetes adversely impacts many organ systems including the skeleton. Clinical trials have revealed a startling elevation in fracture risk in diabetic patients. Bone fractures can be life threatening: nearly 1 in 6 hip fracture patients die within one year. Because physical exercise is proven to improve bone properties and reduce fracture risk in non-diabetic subjects, we tested its efficacy in type 1 diabetes. We hypothesized that diabetic bone's response to anabolic mechanical loading would be attenuated, partially due to impaired mechanosensing of osteocytes under hyperglycemia. Heterozygous C57BL/6-Ins2^Akita/J (Akita) male and female diabetic mice and their age- and gender-matched wild-type (WT) C57BL/6J controls (7-month-old, N = 5–7 mice/group) were subjected to unilateral axial ulnar loading with a peak strain of 3500 με at 2 Hz and 3 min/day for 5 days. The Akita female mice, which exhibited a relatively normal body weight and a mild 40% elevation of blood glucose level, responded with increased bone formation (+ 6.5% in Ct.B.Ar, and 4 to 36-fold increase in Ec.BFR/BS and Ps.BFR/BS), and the loading effects, in terms of changes of static and dynamic indices, did not differ between Akita and WT females (p ≥ 0.1). However, loading-induced anabolic effects were greatly diminished in Akita males, which exhibited reduced body weight, severe hyperglycemia (+ 230%), diminished bone formation (ΔCt.B.Ar: 0.003 vs. 0.030 mm², p = 0.005), and suppressed periosteal bone appositions (ΔPs.BFR/BS, p = 0.02). Hyperglycemia (25 mM glucose) was further found to impair the flow-induced intracellular calcium signaling in MLO-Y4 osteocytes, and significantly inhibited the flow-induced downstream responses including reduction in apoptosis and sRANKL secretion and PGE₂ release. These results, along with previous findings showing adverse effects of hyperglycemia on osteoblasts and mesenchymal stem cells, suggest that failure to maintain normal glucose levels may impair bone's responses to mechanical loading in diabetics.     

Aging diminishes lamellar and woven bone formation induced by tibial compression in adult C57BL/6

Aging purportedly diminishes the ability of the skeleton to respond to mechanical loading, but recent data show that old age did not impair loading-induced accrual of bone in BALB/c mice. Here, we hypothesized that aging limits the response of the tibia to axial compression over a range of adult ages in the commonly used C57BL/6. We subjected the right tibia of old (22 month), middle-aged (12 month) and young-adult (5 month) female C57BL/6 mice to peak periosteal strains (measured near the mid-diaphysis) of − 2200 με and − 3000 με (n = 12–15/age/strain) via axial tibial compression (4 Hz, 1200 cycles/day, 5 days/week, 2 weeks). The left tibia served as a non-loaded, contralateral control. In mice of every age, tibial compression that engendered a peak strain of − 2200 με did not alter cortical bone volume but loading to a peak strain of − 3000 με increased cortical bone volume due in part to woven bone formation. Both loading magnitudes increased total volume, medullary volume and periosteal bone formation parameters (MS/BS, BFR/BS) near the cortical midshaft. Compared to the increase in total volume and bone formation parameters of 5-month mice, increases were less in 12- and 22-month mice by 45–63%. Moreover, woven bone incidence was greatest in 5-month mice. Similarly, tibial loading at − 3000 με increased trabecular BV/TV of 5-month mice by 18% (from 0.085 mm³/mm³), but trabecular BV/TV did not change in 12- or 22-month mice, perhaps due to low initial BV/TV (0.032 and 0.038 mm³/mm³, respectively). In conclusion, these data show that while young-adult C57BL/6 mice had greater periosteal bone formation following loading than middle-aged or old mice, aging did not eliminate the ability of the tibia to accrue cortical bone.     

Altered distributions of bone tissue mineral and collagen properties in women with fragility fractures

Heterogeneity of bone tissue properties is emerging as a potential indicator of altered bone quality in pathologic tissue. The objective of this study was to compare the distributions of tissue properties in women with and without histories of fragility fractures using Fourier transform infrared (FTIR) imaging. We extended a prior study that examined the relationship of the mean FTIR properties to fracture risk by analyzing in detail the widths and the tails of the distributions of FTIR properties in biopsies from fracture and non-fracture cohorts. The mineral and matrix properties of cortical and trabecular iliac crest tissue were compared in biopsies from women with a history of fragility fracture (+ Fx; n = 21, age: mean 54 ± SD 15 y) and with no history of fragility fracture (− Fx; n = 12, age: 57 ± 5 y). A subset of the patients included in the − Fx group were taking estrogen-plus-progestin hormone replacement therapy (HRT) (− Fx + HRT n = 8, age: 58 ± 5 y) and were analyzed separately from patients with no history of HRT (− Fx − HRT n = 4, age: 56 ± 7 y). When the FTIR parameter mean values were examined by treatment group, the trabecular tissue of − Fx − HRT patients had a lower mineral:matrix ratio (M:M) and collagen maturity (XLR) than that of − Fx + HRT patients (− 22% M:M, − 18% XLR) and + Fx patients (− 17% M:M, − 18% XLR). Across multiple FTIR parameters, tissue from the − Fx − HRT group had smaller low-tail (5th percentile) values than that from the − Fx + HRT or + Fx groups. In trabecular collagen maturity and crystallinity (XST), the − Fx − HRT group had smaller low-tail values than those in the –Fx + HRT group (− 16% XLR, − 5% XST) and the + Fx group (− 17% XLR, − 7% XST). The relatively low values of trabecular mineral:matrix ratio and collagen maturity and smaller low-tail values of collagen maturity and crystallinity observed in the − Fx − HRT group are characteristic of younger tissue. Taken together, our data suggest that the presence of newly formed tissue that includes small/imperfect crystals and immature crosslinks, as well as moderately mature tissue, is an important characteristic of healthy, fracture-resistant bone. Finally, the larger mean and low-tail values of mineral:matrix ratio and collagen maturity noted in our − Fx + HRT vs. − Fx − HRT biopsies are consistent with greater tissue age and greater BMD arising from decreased osteoclastic resorption in HRT-treated patients.     

Skin wound trauma,following high-dose radiation exposure,amplifies and prolongs skeletal tissue loss

The present study investigated the detrimental effects of non-lethal, high-dose (whole body) γ-irradiation on bone, and the impact that radiation combined with skin trauma (i.e. combined injury) has on long-term skeletal tissue health. Recovery of bone after an acute dose of radiation (RI; 8 Gy), skin wounding (15–20% of total body skin surface), or combined injury (RI + Wound; CI) was determined 3, 7, 30, and 120 days post-irradiation in female B6D2F1 mice and compared to non-irradiated mice (SHAM) at each time-point. CI mice demonstrated long-term (day 120) elevations in serum TRAP 5b (osteoclast number) and sclerostin (bone formation inhibitor), and suppression of osteocalcin levels through 30 days as compared to SHAM (p < 0.05). Radiation-induced reductions in distal femur trabecular bone volume fraction and trabecular number through 120 days post-exposure were significantly greater than non-irradiated mice (p < 0.05) and were exacerbated in CI mice by day 30 (p < 0.05). Negative alterations in trabecular bone microarchitecture were coupled with extended reductions in cancellous bone formation rate in both RI and CI mice as compared to Sham (p < 0.05). Increased osteoclast surface in CI animals was observed for 3 days after irradiation and remained elevated through 120 days (p < 0.01). These results demonstrate a long-term, exacerbated response of bone to radiation when coupled with non-lethal wound trauma. Changes in cancellous bone after combined trauma were derived from extended reductions in osteoblast-driven bone formation and increases in osteoclast activity.     

TBS and BMD at the end of AI-therapy: A prospective study of the B-ABLE cohort

IntroductionPatients with breast cancer under aromatase inhibitor (AI) treatment often develop osteoporosis and their average bone loss rate is twice that of natural reduction during menopause, increasing fracture risk. As the current diagnostic technique based on bone mineral density (BMD) provides no information on bone quality, the Trabecular Bone Score (TBS) has been proposed to reflect bone microarchitecture status. The present study was designed to assess prospective changes in TBS and lumbar spine (LS) BMD in postmenopausal women with breast cancer at completion of AI treatment.MethodsB-ABLE is a prospective cohort of 735 women with breast cancer treated with AIs according to American Society of Clinical Oncology recommendations: 5 years of AI starting within 6 weeks post-surgery or 1 month after the last cycle of chemotherapy (5y-AI group), or switching to an AI to complete 5-year therapy after 2–3 years of tamoxifen (pTMX-AI group). Patients with osteoporosis were treated with oral bisphosphonates (BP). TBS and LS-BMD changes at completion of AI therapy were evaluated by Student t-test for paired samples. Pearson correlation coefficients were computed for correlations between LS-BMD and TBS.ResultsAI treatment was completed by 277 women. Of these, 70 (25.3%) were allocated to BP therapy. The non-BP-treated patients (74.7%) showed significant decreases in TBS (− 2.94% in pTMX-AI and − 2.93% in 5y-AI groups) and in LS-BMD (− 4.14% in pTMX-AI and − 2.28% in 5y-AI groups) at the end of AI treatment. In BP-treated patients, TBS remained stable at the end of AI treatment, whereas LS-BMD showed significant increases (+ 2.30% in pTMX-AI and + 5.33% in 5y-AI groups). Moderate associations between TBS and LS-BMD values at baseline and at the end of AI treatment (r = 0.4; P < 0.001) were observed. At the end of treatment, changes in spine BMD and TBS were weakly correlated (r = 0.1, P < 0.01).ConclusionsAI therapy induces significant decreases in TBS, comparable to BMD loss. BP-treated patients maintained TBS values, whereas BMD increased. AI treatment leads to deterioration of bone microarchitecture, which seems to be attenuated by BP therapy.