The Effects of Acute Binge Alcohol Consumption on the Trabecular Morphometry and Tensile Strength of Adolescent Sprague Dawley Rat Femora

SUMMARY: Excessive alcohol consumption adversely affects bone metabolism, thus resulting in reduced bone length, density, and strength. Moreover, these deficits in bone density and strength are likely to increase the risk of fragility fractures and the early onset of osteoporosis. While excessive alcohol consumption is an established risk factor for osteoporotic fractures, there remains a dearth of information in literature about bone effects of binge alcohol consumption in adolescents. Therefore, our study aimed to examine the effects of acute binge alcohol consumption on the adolescent bone micro-architecture and tensile strength. Twelve male Sprague Dawley rats aged 7 weeks were randomly placed in 2 groups: alcohol (n =6), receiving alcohol (3g/kg) and pair-fed control (n = 6), receiving an isocaloric equivalent of maltose dextrin via oral gavage for 3 days in one week (on alternative days). The femora were dissected and scanned using a Micro-Focus X-ray Computed Tomography (3D-µCT). Following reconstruction, trabecular morphometry was assessed in both the proximal and distal epiphysis, using a Volume Graphics Studio® software. A three-point bending test was employed to examine the effect of alcohol on the tensile strength of the bone. Results showed trabeculae parameters to be affected in the distal epiphysis of the femur, while in the proximal epiphysis it remained unaffected. Tensile strength parameters were also not affected by the consumption of alcohol. These findings may suggest that acute binge alcohol consumption has detrimental effects on the bone micro-architecture specific to the distal epiphysis.

El consumo excesivo de alcohol afecta negativamente al metabolismo óseo, lo que resulta en una reducción de la longitud, densidad y resistencia de los huesos. Además, es probable que estos déficits en la densidad y la fuerza ósea aumenten el riesgo de fracturas por fragilidad y la aparición temprana de osteoporosis. Si bien el consumo excesivo de alcohol es un factor de riesgo establecido para las fracturas osteoporóticas, existe escasa información en la literatura sobre los efectos óseos del consumo excesivo de alcohol en adolescentes. Por lo tanto, nuestro estudio tuvo como objetivo examinar los efectos del consumo excesivo de alcohol en la microarquitectura ósea y la resistencia a la tracción e n ratas adolescentes. Doce ratas macho Sprague Dawley de 7 semanas de edad se colocaron aleatoriamente en 2 grupos: alcohol (n = 6), que recibieron alcohol (3 g/kg) y control (n = 6), que recibieron un equivalente isocalórico de maltosa dextrina mediante sonda oral, durante 3 días en una semana (en días alternos). Los fémures se diseccionaron y escanearon mediante una tomografía computarizada de rayos X con microenfoque (3D-mCT). Después de la reconstrucción, se evaluó la morfometría trabecular tanto en la epífisis proximal como en la distal, utilizando un software Volume Graphics Studio®. Se empleó una prueba de flexión de tres puntos para examinar el efecto del alcohol sobre la resistencia a la tracción del hueso. Los resultados mostraron que los parámetros de las trabéculas se vieron afectados en la epífisis distal del fémur, mientras que en la epífisis proximal no se observaron afectados. Los parámetros de resistencia a la tracción tampoco se vieron afectados por el consumo de alcohol. Estos hallazgos pueden sugerir que el consumo excesivo de alcohol tiene efectos perjudiciales sobre la microarquitectura ósea específica de la epífisis distal del hueso.

Ethanolic extract of Pyrrosia lingua (Thunb.) Farw. ameliorates OVX-induced bone loss and RANKL-induced osteoclastogenesis.

Pyrrosia lingua (Thunb.) Farw is a common plant that has been widely used as a traditional herbal medicine in China and Korea to treat patients suffering from pain, vaginal bleeding and urolithiasis. However, the pharmacological effects of P. lingua on bone remain unknown. We investigated the anti-osteoporotic effects of an ethanolic extract of P. lingua (EEPL). We found that EEPL suppressed osteoclast differentiation by directly acting on osteoclast precursor cells. EEPL suppressed the expression of receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor of activated T cells 1, a major transcription factor for osteoclastogenesis, by inhibiting RANKL-induced expression of aryl hydrocarbon receptor/c-Fos, and activation of nuclear factor-κB and mitogen-activated protein kinases. Moreover, administration of EEPL inhibited trabecular bone loss and weight gain in ovariectomized mice. Furthermore, we identified phytochemicals in EEPL that are known to exert anti-osteoclastogenic or anti-osteoporotic effects using ultra-high-performance liquid chromatography-tandem mass-spectrometry analysis. Overall, the results of this study suggest that EEPL is effective therapeutic candidate that can be used to prevent or treat postmenopausal osteoporosis.

The Influence of Prenatal Fumonisin Exposure on Bone Properties, as well as OPG and RANKL Expression and Immunolocalization, in Newborn Offspring Is Sex and Dose Dependent.

The current study examined the effects of exposure of pregnant dams to fumonisins (FBs; FB1 and FB2), from the seventh day of pregnancy to parturition, on offspring bone metabolism and properties. The rats were randomly divided into three groups intoxicated with FBs at either 0, 60, or 90 mg/kg b.w. Body weight and bone length were affected by fumonisin exposure, irrespective of sex or dose, while the negative and harmful effects of maternal FBs' exposure on bone mechanical resistance were sex and dose dependent. The immunolocalization of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL), in bone and articular cartilage, indicated that the observed bone effects resulted from the FB-induced alterations in bone metabolism, which were confirmed by the changes observed in the Western blot expression of OPG and RANKL. It was concluded that the negative effects of prenatal FB exposure on the general growth and morphometry of the offspring bones, as a result of the altered expression of proteins responsible for bone metabolism, were dose and sex dependent.

Additive Manufacturing of Caffeic Acid-Inspired Mineral Trioxide Aggregate/Poly-ε-Caprolactone Scaffold for Regulating Vascular Induction and Osteogenic Regeneration of Dental Pulp Stem Cells.

Mineral trioxide aggregate (MTA) is a common biomaterial used in endodontics regeneration due to its antibacterial properties, good biocompatibility and high bioactivity. Surface modification technology allows us to endow biomaterials with the necessary biological targets for activation of specific downstream functions such as promoting angiogenesis and osteogenesis. In this study, we used caffeic acid (CA)-coated MTA/polycaprolactone (PCL) composites and fabricated 3D scaffolds to evaluate the influence on the physicochemical and biological aspects of CA-coated MTA scaffolds. As seen from the results, modification of CA does not change the original structural characteristics of MTA, thus allowing us to retain the properties of MTA. CA-coated MTA scaffolds were shown to have 25% to 55% higher results than bare scaffold. In addition, CA-coated MTA scaffolds were able to significantly adsorb more vascular endothelial growth factors (p < 0.05) secreted from human dental pulp stem cells (hDPSCs). More importantly, CA-coated MTA scaffolds not only promoted the adhesion and proliferation behaviors of hDPSCs, but also enhanced angiogenesis and osteogenesis. Finally, CA-coated MTA scaffolds led to enhanced subsequent in vivo bone regeneration of the femur of rabbits, which was confirmed using micro-computed tomography and histological staining. Taken together, CA can be used as a potently functional bioactive coating for various scaffolds in bone tissue engineering and other biomedical applications in the future.

Trabecular Bone Parameters, TIMP-2, MMP-8, MMP-13, VEGF Expression and Immunolocalization in Bone and Cartilage in Newborn Offspring Prenatally Exposed to Fumonisins.

Fumonisins are protein serine/threonine phosphatase inhibitors and potent inhibitors of sphingosine N-acyltransferase (ceramide synthase) disrupting de novo sphingolipid biosynthesis. The experiment was conducted to evaluate the effects of fumonisins (FB) exposure from the 7th day of pregnancy to parturition on offspring bone development. The rats were randomly allocated to either a control group (n = 6), not treated with FBs, or to one of the two groups intoxicated with FBs (either at 60 mg FB/kg b.w. or at 90 mg FB/kg b.w. Numerous negative, offspring sex-dependent effects of maternal FB exposure were observed with regards to the histomorphometry of trabecular bone. These effects were due to FB-inducted alterations in bone metabolism, as indicated by changes in the expression of selected proteins involved in bone development: tissue inhibitor of metalloproteinases 2 (TIMP-2), matrix metalloproteinase 8 (MMP-8), matrix metalloproteinase 13 (MMP-13), and vascular endothelial growth factor (VEGF). The immunolocalization of MMPs and TIMP-2 was performed in trabecular and compact bone, as well as articular and growth plate cartilages. Based on the results, it can be concluded that the exposure of pregnant dams to FB negatively affected the expression of certain proteins responsible for bone matrix degradation in newborns prenatally exposed to FB in a dose- and sex-dependent manner.

Tributyltin protects against ovariectomy-induced trabecular bone loss in C57BL/6J mice with an attenuated effect in high fat fed mice.

Risk factors for poor bone quality include estrogen loss at menopause, a high fat diet and exposures to drugs/chemicals that activate peroxisome proliferator activated receptor gamma (PPARγ). We previously reported that the PPARγ and retinoid X receptor dual ligand, tributyltin (TBT), repressed periosteal bone formation but enhanced trabecular bone formation in vivo. Here, we examined the interaction of diet, ovariectomy (OVX) and TBT exposure on bone structure. C57BL/6J mice underwent either sham surgery or OVX at 10 weeks of age. At 12 weeks of age, they were placed on a low (10% kcal) or high (45% kcal) fat, sucrose-matched diet and treated with vehicle or TBT (1 or 5 mg/kg) for 14 weeks. OVX increased body weight gain in mice on either diet. TBT enhanced body weight gain in intact mice fed a high fat diet, but decreased weight gain in OVX mice. Elemental tin concentrations increased dose-dependently in bone. TBT had marginal effects on cortical and trabecular bone in intact mice fed either diet. OVX caused a reduction in cortical and trabecular bone, regardless of diet. In high fat fed OVX mice, TBT further reduced cortical thickness, bone area and total area. Interestingly, TBT protected against OVX-induced trabecular bone loss in low fat fed mice. The protective effect of TBT was nullified by the high fat. These results show that TBT protects against trabecular bone loss, even in the presence of a strongly resorptive environment, at an even lower level of exposure than we showed repressed homeostatic resorption.

Intrauterine alcohol exposure disturbs trabecular morphology in the Sprague Dawley rat humerus epiphysis up to 3- weeks postnatally / La exposición al alcohol intrauterino altera la morfología trabecular en la epífisis del húmero de la rata Sprague Dawley hasta 3 semanas después del nacimiento

SUMMARY: Gestational alcohol exposure inhibits neurological as well as bone growth and development both in fetal and postnatal life. Stunted stature, osteoporosis and fractures in adult life are some of the adverse effects. While the impact of intrauterine alcohol on the brain has been extensively investigated, studies on the effects on bone are relatively few. Therefore, our study aimed to examine the impact of prenatal alcohol exposure on bone microarchitecture in 3-week-old rats using Micro-focus X-Ray Computed Tomography (Micro CT). Time mated pregnant Sprague Dawley dams (13) were randomly placed into 3 groups: ethanol (n=5), saline control (n=5) and untreated control (n=3). The former 2 groups received treatment with 0.015ml/g of 25.2 % ethanol and 0.9 % saline, respectively, for the first 19 days of gestation. The untreated group received no treatment. The pups remained with their dams until termination at 21 days of age. From each dam, 2 pups were collected resulting in: ethanol (n=10), saline controls (n= 10) and untreated controls (n = 6). The humeri of the pups were dissected and scanned using a 3D-μCT scanner (Nikon XTH 225L) at 15μm resolution. Trabecular and cortical parameters were analysed using Volume Graphics Studio® software following reconstruction. Results showed a decrease in trabecular size, spaces, thickness, and volume. There was a decrease in cortical bone area in the ethanol group compared to the controls. These findings may suggest that osteoporosis and fractures seen as gestational alcohol effects may be due to compromised trabecular structure.

RESUMEN: La exposición al alcohol durante la gestación inhibe el crecimiento y desarrollo neurológico y óseo tanto en la vida fetal como posnatal. Algunos de los efectos adversos incluyen la estatura atrofiada, osteoporosis y fracturas en la vida adulta. Si bien se ha estudiado el impacto del alcohol intrauterino en el cerebro, los estudios sobre los efectos en los huesos son escasos. Por lo tanto, nuestro estudio tuvo como objetivo examinar el impacto de la exposición prenatal al alcohol en la microarquitectura ósea en ratas de 3 semanas de edad utilizando Tomografía Computarizada de Rayos X Micro-focus (Micro CT). Las hembras de Sprague Dawley preñadas con apareamiento temporal (13) se colocaron aleatoriamente en 3 grupos: etanol (n = 5), control de solución salina (n = 5) y control sin tratar (n = 3). Los primeros 2 grupos recibieron tratamiento con 0,015 ml /g de etanol al 25,2 % y solución salina al 0,9 %, respectivamente, durante los primeros 19 días de gestación. El grupo no tratado no recibió tratamiento. Las crías permanecieron con sus madres hasta la terminación a los 21 días de edad. De cada madre, se recolectaron 2 crías que dieron como resultado: etanol (n = 10), controles salinos (n = 10) y controles no tratados (n = 6). Se diseccionaron y escanearon los húmero de las crías usando un escáner 3D-μCT (Nikon XTH 225L) a una resolución de 15 μm. Los parámetros trabeculares y corticales se analizaron utilizando el software Volume Graphics Studio® después de la reconstrucción. Los resultados mostraron una disminución en el tamaño trabecular, los espacios, el grosor y el volumen. Hubo una disminución en el área del hueso cortical en el grupo de etanol en comparación con los controles. Estos hallazgos pueden sugerir que la osteoporosis y las fracturas por causa de los efectos del alcohol gestacional se pueden deber a una estructura trabecular comprometida.

Impact of bone-active drugs and underlying disease on bone health after lung transplantation: A longitudinal study.

INTRODUCTION: the effect of bone-active drugs on the risk of fragility fractures (Fx), bone mineral density (BMD) and trabecular bone score (TBS) changes in patients receiving lung transplantation (LTx) is largely unknown. This study assessed the bone-active drugs effect in patients undergoing LTx both with (CF) and without (nCF) cystic-fibrosis. METHODS: We evaluated incident Fx, both clinical and morphometric vertebral Fx by spinal X-ray, BMD and trabecular bone score (TBS) in 117 patients (CF=50, nCF n = 67) before and 24-months after LTx. A bone-active therapy was proposed to all LTx candidates. RESULTS: 83.8% of patients started a bone-active drug. Lumbar-spine (LS) T-score improved significantly only in treated patients (-1.4 ± 1.0 vs -2.0±1.0, p = 0.0001), whereas femur BMD and TBS remained stable in treated and not treated subjects. The rate of incident Fx was 15.3%, with no difference between treated and not treated patients. After LTx, LS T-score improved significantly only in nCF group (-1.3 ± 1.0 vs -1.8 ± 1.1, p = 0.0001), while femur remained stable in both nCF and CF groups. Patients with CF showed a significant Z-TBS increase (-3.6 ± 1.7 vs -3.0 ± 1.7, p = 0.019) and a lower Fx incidence as compared with nCF patients (4.1% vs 24.2%, p  =0.003). Incident Fx were associated with nCF diagnosis (OR 7.300, CI95% 1.385-38.461, p = 0.019) regardless of prevalent Fx, previous glucocorticoid therapy and bone-active therapy introduced at least 6 months before LTx. CONCLUSIONS: A prompt medical intervention helps in preventing BMD loss after LTx. As compared with nCF patients, CF patients show a TBS increase and a lower Fx risk after LTx.

Inhibition of miR-29 Activity in the Myeloid Lineage Increases Response to Calcitonin and Trabecular Bone Volume in Mice.

The miR-29-3p family (miR-29a, miR-29b, miR-29c) of microRNAs is increased during receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclastogenesis. In vivo, activation of a miR-29-3p tough decoy inhibitor in Cre recombinase under the control of the lysozyme 2 promoter-expressing cells (myeloid lineage) resulted in mice displaying enhanced trabecular and cortical bone volume because of decreased bone resorption. Calcitonin receptor (Calcr) is a miR-29 target that negatively regulates bone resorption. CALCR was significantly increased in RANKL-treated miR-29-decoy osteoclasts, and these cells were more responsive to the inhibitory effect of calcitonin on osteoclast formation. Further, cathepsin K (Ctsk), which is critical for resorption, was decreased in miR-29-decoy cells. CALCR is a Gs-coupled receptor and its activation raises cAMP levels. In turn, cAMP suppresses cathepsin K, and cAMP levels were increased in miR-29-decoy cells. siRNA-mediated knock-down of Calcr in miR-29 decoy osteoclasts allowed recovery of cathepsin K levels in these cells. Overall, using a novel knockin tough decoy mouse model, we identified a new role for miR-29-3p in bone homeostasis. In RANKL-driven osteoclastogenesis, as seen in normal bone remodeling, miR-29-3p promotes resorption. Consequently, inhibition of miR-29-3p activity in the myeloid lineage leads to increased trabecular and cortical bone. Further, this study documents an interrelationship between CALCR and CTSK in osteoclastic bone resorption, which is modulated by miR-29-3p.

Intermittent administration sodium valproate has a protective effect on bone health in ovariectomized rats.

The present work was aimed to evaluate the effect of different administration modes of sodium valproate (VPA) on bone strength, bone mass and bone mineral density in ovariectomized (OVX) rats and further investigation of the possible mechanism. 60 female SD rats were randomly divided into 4 groups: Sham group (Sham, n = 15), OVX group (OVX, n = 15), OVX rats received intermittent VPA treatment group (IVPA, n = 15) and OVX rats received daily VPA treatment group (EVPA, n = 15). After 12 weeks of treatment, the rats were sacrificed, and serum and femur samples were harvested. DEXA, Micro-CT, history, biomechanical testing, biochemical index and western blot analysis were used to observe the therapeutic effect and explore the possible mechanism. Micro-CT and DEXA analysis of bones revealed better BMD and higher BV/TV, Tb. Th, Tb. N, Conn. D and lower Tb. Sp at femoral metaphysis evaluated in IVPA when compared with OVX and EVPA group (P < 0.05). Histological, fluorescent analysis and biological strength revealed more trabecular bone and higher relative mineral apposition rate, maximal load, elastic modulus and energy at break with evaluated in IVPA when compared with OVX and EVPA group (P < 0.05). The levels of P1NP, estrogen, CTX, TRAP-5b and RANKL of the IVPA group showed a significant increase when compared with the OVX and EVPA group (P < 0.05). We confirm adverse effects on protein expressions including Notch1, Jagged1, HEY1, Wnt 1, ß-catenin and RUNX2 following daily VPA treatment in OVX female rats. Our current study demonstrated that intermittent administration of sodium valproate has a protective effect on bone health in OVX rats and these effects may be achieved by activating Notch/Wnt/ß-catenin/RUNX2 signal axis.

The effect of parathyroid hormone on osteogenesis is mediated partly by osteolectin.

We previously described a new osteogenic growth factor, osteolectin/Clec11a, which is required for the maintenance of skeletal bone mass during adulthood. Osteolectin binds to Integrin α11 (Itga11), promoting Wnt pathway activation and osteogenic differentiation by leptin receptor+ (LepR+) stromal cells in the bone marrow. Parathyroid hormone (PTH) and sclerostin inhibitor (SOSTi) are bone anabolic agents that are administered to patients with osteoporosis. Here we tested whether osteolectin mediates the effects of PTH or SOSTi on bone formation. We discovered that PTH promoted Osteolectin expression by bone marrow stromal cells within hours of administration and that PTH treatment increased serum osteolectin levels in mice and humans. Osteolectin deficiency in mice attenuated Wnt pathway activation by PTH in bone marrow stromal cells and reduced the osteogenic response to PTH in vitro and in vivo. In contrast, SOSTi did not affect serum osteolectin levels and osteolectin was not required for SOSTi-induced bone formation. Combined administration of osteolectin and PTH, but not osteolectin and SOSTi, additively increased bone volume. PTH thus promotes osteolectin expression and osteolectin mediates part of the effect of PTH on bone formation.

Solanum nigrum Line inhibits osteoclast differentiation and suppresses bone mineral density reduction in the ovariectomy­induced osteoporosis model.

Bone homeostasis is maintained by osteoclasts that absorb bone and osteoblasts that form bone tissue. Menopausal osteoporosis is a disease associated with aging and hormonal changes due to menopause causing abnormal activation of osteoclasts, resulting in a decrease in bone density. Existing treatments for osteoporosis have been reported to have serious side effects, such as jawbone necrosis and breast and uterine cancer; therefore, their use by patients is decreasing, whilst studies focusing on alternative treatments are increasingly popular. Solanum nigrum Line (SL) has been used as a medicinal plant that possesses several pharmacological effects, such as anti­inflammatory and hepatotoxic protective effects. To the best of our knowledge, however, its effects on osteoporosis and osteoclasts have not been demonstrated previously. In the present study, the anti­osteoporotic effect of SL was investigated using a postmenopausal model of osteoporosis in which Sprague­Dawley rat ovaries were extracted. In addition, the inhibitory effects on osteoclast differentiation and function of SL was confirmed using an osteoclast model treated with receptor activator of NF­κB ligand (RANKL) on murine RAW 264.7 macrophages. In vivo experiments showed that SL reduced the decrease in bone mineral density and improved changes in the morphological index of bone microstructure, such as trabecular number and separation. In addition, the number of tartrate resistant acid phosphatase­positive cells in the femur and the expression levels of nuclear factor of activated T­cells cytoplasmic 1 (NFATc1) and cathepsin K protein were inhibited. In vitro, SL suppressed RANKL­induced osteoclast differentiation and bone resorption ability; this was mediated by NFATc1/c­Fos, a key transcription factor involved in osteoclast differentiation, ultimately inhibiting expression of various osteoclast­associated genes. These experimental results show that SL may be an alternative treatment for osteoporosis caused by abnormal activation of osteoclasts in the future.

Trabecular bone score in women with differentiated thyroid cancer on long-term TSH-suppressive therapy.

INTRODUCTION: Thyrotropin stimulating hormone (TSH) suppression in patients with differentiated thyroid cancer (DTC) aims to decrease the growth and proliferation of thyroid cancer cells. However, the effect of TSH-suppressive therapy on bone microarchitecture remains undefined. METHODS: Cross-sectional study including 43 women with DTC undergoing TSH-suppressive therapy (sTSH) compared to 20 women also on levothyroxine (LT4) therapy but with TSH in the low-normal range (nTSH) since the thyroid surgery. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DXA), and trabecular bone score (TBS) was evaluated using the TBS iNsigth software. Fracture risk assessed by FRAX, with and without TBS, was calculated. The relationship between suppressive therapy-related parameters and bone parameters was investigated. RESULTS: The TBS mean values were not significantly different in the sTSH and nTSH groups (1.273 ± 0.12 vs 1.307 ± 0.14, p = 0.7197). In both groups, postmenopausal women had degraded microarchitecture (TBS 1.216 ± 0.11 vs 1.213 ± 0.09, p = 0.9333), while premenopausal women had normal microarchitecture (1.328 ± 0.11 vs 1.401 ± 0.12, p = 0.195). The percentage of all postmenopausal women with degraded TBS was 54.7%, while the percentage of osteoporosis diagnoses was 16.1%. The TBS-adjusted FRAX-probability of fracture was similar in sTSH and nTSH groups. Body mass index (BMI) and menopausal status were the only variables associated with TBS and BMD. CONCLUSION: Trabecular microarchitecture assessed by TBS was similar between women on long-term suppressive therapy in DTC and those on LT4 replacement therapy aiming at a TSH level within the low-normal reference range. Low TBS values were observed in postmenopausal women of both groups, suggesting that not only suppressed TSH levels but also a low-normal TSH is associated with deteriorated bone microarchitecture in postmenopausal women following total thyroidectomy.

Intramedullary injury combined with osteoporosis therapeutics regulates targeted local osteogenesis.

Bone marrow ablation prompts transient bone formation in nearly the entire medullary cavity before marrow regeneration occurs. Here, we establish a procedure to direct bone formation in a desired particular site within the medullary cavity for support of biomedical devices. Local intramedullary injury was performed in the tibiae of rats and parathyroid hormone (PTH), alendronate, or saline was administered. Newly generated bone in the medulla was assessed by micro-CT and histology. To evaluate the function of newly generated bone, animals received intramedullary injury in tibiae followed by daily PTH. At day-14, implants were placed in the endocortical bone and the bone response to the implants was assessed. The fate of newly generated bone was compared with and without implants. We found that neither intramedullary injury nor medication alone resulted in bone formation. However, when combined, substantial bone was generated locally inside the diaphyseal medulla. Newly formed bone disappeared without implant placement but was retained with implants. Bone was especially retained around and between the implants. This study found that local bone marrow disruption followed by PTH or alendronate generated substantial cancellous bone locally in the diaphyseal medulla. This approach offers promise as a tissue engineering tool in medicine and dentistry.

PERK activator CCT020312 prevents inflammation-mediated osteoporosis in the ovariectomized rats.

OBJECTIVE: To investigate the therapeutic effects of PERK activator CCT020312 (CCT) on inflammation-mediated osteoporosis (IMO) in ovariectomized rats. METHODS: Rats were divided into Sham, IMO, IMO + 1 mg/kg CCT and IMO + 2 mg/kg CCT groups. IMO models were constructed by bilateral ovariectomy (OVX) on 1st day followed by injection with magnesium silicate (Talc) on the 59th day. Sham rats did not undergo OVX surgery and were injected with saline instead of Talc. From 60th to 79th day, rats were treated with DMSO (vehicle control) in the Sham and IMO groups, and 1 or 2 mg/kg CCT020312 in treatment groups. Osteopontin (OPN), osteocalcin (OCN), tartrate-resistant acid phosphatase (TRAP), C-terminal telopeptide of type I collagen (CTX-I), and pro-inflammatory factors were measured on the 80th day. ProdigyDEXA was used to evaluate bone mineral density and content (BMD/BMC). Bone volume/total volume (BV/TV), connectivity density (Conn.D), trabecular number (Tb.N), and trabecular separation (Tb.Sp) was assessed using 3D micro-CT scanner. RESULTS: CCT up-regulated Conn.D, BV/TV, and Tb.N, but down-regulated Tb.Sp in IMO rats. Besides, the declined femoral BMD and BMC in IMO rats were elevated after CCT treatment. Besides, IMO rats represented declined OPN and OCN, as well as increased TRAP, CTX-I, and pro-inflammatory factors, whereas those in the treatment groups were ameliorated regarding these indexes, with 2 mg/kg CCT showing better effect. CONCLUSION: PERK activator CCT020312 can be served as a new therapeutic option for the protection against bone loss in the OVX rat model associated with inflammation probably by manipulating inflammatory factors.

Therapeutic potential of russelioside B as anti-arthritic agent in Freund's adjuvant-induced arthritis in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Caralluma species are traditional edible herbs used in folkloric medicine as antidiabetic, antioxidant, antipyretic, antirheumatic, anti-inflammatory and anthelmintic agents. C. quadrangula was selected in this study to document the traditional use of the genus as anti-rheumatic treatment and the possible mechanisms of action. AIM OF THE STUDY: The higher mortality rates and shorter survival among the patients suffering from rheumatoid arthritis (RA) led to the increased interest on searching for new treatments for RA. Russelioside B (RB), a major pregnane glycoside found in C. quadrangula, was evaluated as a new anti-rheumatic agent. MATERIALS AND METHODS: The n-butanol fraction of C. quadrangula was chromatographed on a silica gel column to isolate RB. The adjuvant-induced arthritis (AIA) model was established in rats by intradermal injection of complete Freund's adjuvant (CFA) to evaluate its anti-arthritic effect. Ibuprofen was used as a reference drug. Forty rats were randomly divided into 5 groups (n = 8): normal (NOR); CFA model (CFA); ibuprofen, 5 mg/kg; RB, 25 mg/kg and RB, 50 mg/kg. The treatments were initiated from day 16 when AIA model was established and continued up to day 40. Serum diagnostic rheumatoid markers, inflammatory cytokines, oxidative stress biomarkers, cartilage and bone degeneration enzymes were assessed. RESULTS: RB at 50 mg/kg b. wt., showed significant decreases in the activities of hyaluronidase and ß-glucouronidase enzymes as well significant decreases in the levels of proinflammatory cytokines as nuclear factor-kappa-B (NF-κB), tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) compared to the CFA group; 11.04 ± 0.61 pg/mg protein, 4.35 ± 0.25 pg/mg protein, 3.32 ± 0.13 pg/mg protein & 2.75 ± 0.14 pg/mg protein for RB, 50 mg/kg b. wt. group vs. 25.33 ± 2.13 pg/mg protein, 25.65 ± 2.1 pg/mg protein, 22.20 ± 1.34 pg/mg protein & 13.27 ± 1.40 pg/mg protein for the arthritic group, respectively. The total antioxidant capacity (TAC) was significantly restored to normal values in RB, 50 mg/kg treated rats (4.01 ± 0.09 nmol/mL vs. 3.71 ± 0.27 nmol/mL) and the levels of myeloperoxidase (MPO) reduced by 10-folds of the CFA arthritic group. Bone histomorphometry revealed that RB treatment significantly attenuated the CFA-induced bone loss in a dose-dependent manner. CONCLUSIONS: These findings suggested that the anti-arthritic effect of RB was mediated through the reduction of the rheumatoid markers, anti-inflammatory and antioxidant action, inhibition of cartilage and bone degenerative enzymes as well as attenuation of bone loss and osteoclastogenesis.

Evaluation of Bone Marrow Texture and Trabecular Changes With Quantitative DCE-MRI and QCT in Alloxan-Induced Diabetic Rabbit Models.

Purpose: To investigate whether the microvascular permeability of lumbar marrow and bone trabecular changes in early-stage diabetic rabbits can be quantitatively evaluated using dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), quantitative computed tomography, and texture-analyzed permeability parameter Ktrans map of DCE-MRI. Materials and Methods: This prospective study included 24 rabbits that were randomly assigned to diabetic (n = 14) and control (n = 10) groups. All rabbits underwent sagittal MRI of the lumbar region at 0, 4, 8, 12, and 16 weeks after alloxan injection. Pearson correlation coefficient was performed to determine the correlation between permeability parameter and bone mineral density (BMD). Repeated-measures ANOVA was used to analyze the changes in lumbar BMD over time in each group and the texture parameters of diabetic rabbit lumbar marrow at different time points. Mann-Whitney U rank sum test was used to compare the differences of each index between the two groups and calculate the area under the curve (AUC). Results: BMD was correlated with Ktrans , Kep , and Ve but not with Vp . At weeks 0-16, the BMD of the rabbits in the diabetic and normal groups was not statistically significant, but the change in BMD showed an overall downward trend. For texture analysis, entropy, energy, and Uniformized positive pixel (UPP) parameters extracted from the Ktrans map showed significant differences from week 0 to 16 between the two groups. The identification ability at 8-12 weeks was higher than that at 12-16 weeks, and the AUCs were 0.734, 0.766, and 0.734, respectively (P < 0.05 for all). Conclusions: The changes in BMD measured using quantitative computed tomography occurred later than those measured using bone trabecular morphometry. Texture analysis parameters based on DCE-MRI quantitative parameter Ktrans map are feasible to identify early changes in lumbar marrow structure in diabetic rabbits.

Water Extract of Fritillariae thunbergii Bulbus Inhibits RANKL-Mediated Osteoclastogenesis and Ovariectomy-Induced Trabecular Bone Loss.

Fritillariae thunbergii bulbus has been widely used to treat symptoms of coughs and airway congestion in the chest due to pathological colds and damp phlegm in traditional Chinese medicine. Despite its long history of traditional use, its pharmacological activities on osteoclastogenesis and osteoporosis have not been evaluated. This study investigated the effects of the water extract of Fritillariae thunbergii bulbus (WEFT) on osteoclast differentiation in bone marrow-derived macrophage cells and on ovariectomy (OVX)-induced osteoporosis in mice. We found that WEFT significantly inhibited osteoclastogenesis by downregulating the receptor activator of the NF-κB ligand (RANKL) signaling-induced nuclear factor of activated T-cells cytoplasmic 1 (NFATc1) expression. In an OVX-induced osteoporosis model, WEFT significantly prevented the OVX-induced trabecular loss of femurs, accompanied by a reduction in fat accumulation in the bone marrow and liver. In addition, WEFT significantly prevented weight gain and gonadal fat gain without recovering uterine atrophy. Using ultrahigh-performance liquid chromatography-tandem mass spectrometry, seven alkaloids (peimisine glucoside, yibeissine, peiminoside, sipeimine-glucoside, peimisine, peimine, and peiminine) were identified in WEFT. The results of this study suggest that WEFT can be a potential pharmacological candidate to reduce menopausal osteoporosis and menopause-related symptoms, such as fat accumulation.

ß-Catenin signaling is important for osteogenesis and hematopoiesis recovery following methotrexate chemotherapy in rats.

Cancer chemotherapy can significantly impair the bone formation and cause myelosuppression; however, their recovery potentials and mechanisms remain unclear. This study investigated the roles of the ß-catenin signaling pathway in bone and bone marrow recovery potentials in rats treated with antimetabolite methotrexate (MTX) (five once-daily injections, 0.75 mg/kg) with/without ß-catenin inhibitor indocyanine green (ICG)-001 (oral, 200 mg/kg/day). ICG alone reduced trabecular bone volume and bone marrow cellularity. In MTX-treated rats, ICG suppressed bone volume recovery on Day 11 after the first MTX injection. ICG exacerbated MTX-induced decreases on Day 9 osteoblast numbers on bone surfaces, their formation in vitro from bone marrow stromal cells (osteogenic differentiation/mineralization), as well as expression of osteogenesis-related markers Runx2, Osx, and OCN in bone, and it suppressed their subsequent recoveries on Day 11. On the other hand, ICG did not affect MTX-induced increased osteoclast density and the level of the osteoclastogenic signal (RANKL/OPG expression ratio) in bone, suggesting that ICG inhibition of ß-catenin does nothing to abate the increased bone resorption induced by MTX. ICG also attenuated bone marrow cellularity recovery on Day 11, which was associated with the suppressed recovery of CD34+ or c-Kit+  hematopoietic progenitor cell contents. Thus, ß-catenin signaling is important for osteogenesis and hematopoiesis recoveries following MTX chemotherapy.

Cadmium exposure negatively affects the microarchitecture of trabecular bone and decreases the density of a subset of sympathetic nerve fibers innervating the developing rat femur.

Cadmium (Cd) is toxic to the skeletal system resulting in bone loss and pain. We aimed at determining the effect of chronic Cd exposure on bone density and microarchitecture along with changes in the density of a subset of sensory and sympathetic nerve fibers innervating the developing rat femur. Newborn male Wistar rats were injected daily for 49 days with CdCl2 (1 mg/kg i.p.) or saline solution (control group). At the day of sacrifice, levels of Cd in the right femur, liver and kidney were determined by atomic absorption spectrophotometry. Additionally, microCT followed by immunohistochemical analyses were performed in the left femur. Results showed Cd accumulation in trabecular bone neared levels seen in liver and kidney. Cd concentration in cortical bone was significantly lower versus trabecular bone. MicroCT analysis revealed that Cd-exposed rats had a significant decrease in trabecular bone parameters at the distal femoral metaphysis; however, most of the cortical bone parameters were not significantly affected. Cd-exposed rats showed a significant loss of TH+ sympathetic nerve fibers, but not of CGRP+ sensory nerve fibers, at the level of bone marrow of the femoral diaphysis as compared to control rats. This study shows that Cd negatively affects bone density and microarchitecture of trabecular bone and decreases the density of sympathetic nerve fibers innervating rat femur. Future studies are warranted to determine the toxigenic mechanisms of Cd on sympathetic nerves and how sympathetic denervation influences bone loss in animals exposed to Cd.