Improved osteogenesis in rat femur segmental defects treated with human allograft and zinc adjuvants.

Bone allograft is widely used to treat large bone defects or complex fractures. However, processing methods can significantly compromise allograft osteogenic activity. Adjuvants that can restore the osteogenic activity of processed allograft should improve clinical outcomes. In this study, zinc was tested as an adjuvant to increase the osteogenic activity of human allograft in a Rag2 null rat femoral defect model. Femoral defects were treated with human demineralized bone matrix (DBM) mixed with carboxy methyl cellulose containing ZnCl2 (0, 75, 150, 300 µg) or Zn stearate (347 µg). Rat femur defects treated with DBM-ZnCl2 (75 µg) and DBM-Zn stearate (347 µg) showed increased calcified tissue in the defect site compared to DBM alone. Radiograph scoring and µCT (microcomputed tomography) analysis showed an increased amount of bone formation at the defects treated with DBM-Zn stearate. Use of zinc as an adjuvant was also tested using human cancellous bone chips. The bone chips were soaked in ZnCl2 solutions before being added to defect sites. Zn adsorbed onto the chips in a time- and concentration-dependent manner. Rat femur defects treated with Zn-bound bone chips had more new bone in the defects based on µCT and histomorphometric analyses. The results indicate that zinc supplementation of human bone allograft improves allograft osteogenic activity in the rat femur defect model.

Melatonin prevents bone destruction in mice with retinoic acid-induced osteoporosis.

BACKGROUND: The protective effect of melatonin against bone metabolism imbalance in osteoporosis (OP) induced by drugs such as retinoic acid (RA) is unclear. The aim of this study was to explore the role of melatonin in bone destruction based on a mouse model. METHODS: RA-induced OP model mice were established. To assess the effect of melatonin on these mice, micro-CT was used to characterize the trabecular structure of normal mice and those treated with RA (model), RA + low-dose melatonin (Mlt-L), RA + high-dose melatonin (Mlt-H), and RA + alendronate sodium (positive control). The shape of the trabecular bone, the length and diameter of the femoral head and the height and diameter of vertebra(L1) of each group were also measured and the number of osteoclasts was determined by Tartrate-resistant acid phosphatase (TRACP) staining. Meanwhile, the expression of alkaline phosphatase (ALP) was evaluated by immunohistochemistry assays. The differences between groups in terms of liver and kidney oxidation-related indexes and serum and urinary indicators related to bone metabolism were also analyzed. Furthermore, qRT-PCR and western blotting were used to evaluate the effect of melatonin on osteogenic and osteoclastic differentiation in MC3T3-E1 and RAW264.7 cells, respectively. RESULTS: RA induction led to a decrease in the amount and density of trabecular bone, a decrease in the length and diameter of the femur and height, diameter of the vertebra (L1), a decrease in bone mass and density and the expression of ALP, and an increase in the number of osteoclasts. Melatonin treatment alleviated these effects induced by RA, increasing the amount of trabecular bone in OP mice, improving the microstructure of the femur and vertebra(L1) and increasing bone mass bone density and the expression of ALP, as well as decreasing the number of osteoclasts. Additionally, blood and urinary bone metabolism-related indicators showed that melatonin promoted bone formation and inhibited bone resorption. Determination of oxidant and antioxidant biomarkers in the livers and kidneys of the mice revealed that melatonin promoted the antioxidant level and suppressed the level of oxidant molecules in these organs. In vitro, RA promoted osteoclasts and inhibit osteogenesis by increasing oxidative stress levels in the RAW264.7 and MC3T3-E1 cells, but melatonin reversed this effect. Melatonin may, therefore, play a role in the ERK/SMAD and NF-κB pathways. CONCLUSIONS: Melatonin can alleviate bone loss in RA-induced OP model mice, repair the trabecular microstructure, and promote bone formation. These effects may be related to reducing oxidation levels in vivo and vitro through the ERK/SMAD and NF-κB pathways.

Effects of dietary omega-3 fatty acids on bones of healthy mice.

BACKGROUND & AIMS: Altering the lipid component in diets may affect the incidence of metabolic bone disease in patients dependent on parenteral nutrition. Consumption of polyunsaturated fatty acids (PUFA) can impact bone health by modulating calcium metabolism, prostaglandin synthesis, lipid oxidation, osteoblast formation, and osteoclastogenesis. The aim of this study was to evaluate the dietary effects of PUFA on murine bone health. METHODS: Three-weeks-old male (n = 30) and female (n = 30) C57BL/6J mice were randomized into one of three dietary groups. The diets differed only in fat composition: soybean oil (SOY), rich in ω-6 PUFA; docosahexaenoic acid alone (DHA), an ω-3 PUFA; and DHA with arachidonic acid, an ω-6 PUFA, at a 20:1 ratio (DHA/ARA). After 9 weeks of dietary treatment, femurs were harvested for micro-computed tomographic analysis and mechanical testing via 3-point bending. Separate mice from each group were used solely for serial blood draws for measurement of biomarkers of bone formation and resorption. RESULTS: At the microstructural level, although some parameters in cortical bone reached differences that were statistically significant in female mice, these were too small to be considered biologically relevant. Similarly, trabecular bone parameters in male mice were statistically different in some dietary groups, although the biological interpretation of such subtle changes translate into a lack of effect in favor of any of the experimental diets. No differences were noted at the mechanical level and in blood-based biomarkers of bone metabolism across dietary groups within gender. CONCLUSIONS: Subtle differences were noted at the bones' microstructural level, however these are likely the result of random effects that do not translate into changes that are biologically relevant. Similarly, differences were not seen at the mechanical level, nor were they reflected in blood-based biomarkers of bone metabolism. Altogether, dietary consumption of PUFA do not seem to affect bone structure or metabolism in a healthy model of growing mice.

Milk Basic Protein Facilitates Increased Bone Mass in Growing Mice.

Milk basic protein (MBP) comprises a group of basic whey proteins and is effective in preventing bone loss by promoting bone deposition (bone formation) and suppressing withdrawn (bone resorption). We previously revealed the bone protective effects of MBP during life phases involving excessive bone resorption, such as in adults and postmenopausal women, and in animal models (ovariectomized rats and mice). However, it was unclear whether MBP increases bone mass during the growth stage, when there is more bone formation than resorption. We therefore investigated the effect of MBP supplementation on bone mass in 6-wk-old mice provided water supplemented with MBP [0.01%, 0.1%, 1.0% (w/w)] or deionized water (control) ad libitum for 10 wk. Analysis by micro-computerized tomography showed that MBP significantly increased tibia cortical bone mineral density and femur trabecular bone volume to tissue volume compared with mice provided deionized water. Next, the function of MBP in bone remodeling (bone formation and resorption) was evaluated using an in vitro system and the results demonstrated that MBP directly promoted osteoblast proliferation and inhibited osteoclastogenesis. Moreover, the plasma level of insulin-like growth factor-1 was increased by MBP supplementation, suggesting that MBP indirectly promoted osteoblast proliferation/differentiation. These effects enhance bone formation and/or inhibit bone resorption, resulting in increased bone mass in growing mice.

Role of ERαMISS in the Effect of Estradiol on Cancellous and Cortical Femoral Bone in Growing Female Mice

Estrogen receptor-α (ERα) acts primarily in the nucleus as a transcription factor involving two activation functions, AF1 and AF2, but it can also induce membrane-initiated steroid signaling (MISS) through the modulation of various kinase activities and/or secondary messenger levels. Previous work has demonstrated that nuclear ERα is required for the protective effect of the estrogen 17ß-estradiol (E2), whereas the selective activation of ERαMISS is sufficient to confer protection in cortical but not cancellous bone. The aim of this study was to define whether ERαMISS is necessary for the beneficial actions of chronic E2 exposure on bone. We used a mouse model in which ERα membrane localization had been abrogated due to a point mutation of the palmitoylation site of ERα (ERα-C451A). Alterations of the sex hormones in ERα-C451A precluded the interpretation of bone parameters that were thus analyzed on ovariectomized and supplemented or not with E2 (8 µg/kg/d) to circumvent this bias. We found the beneficial action of E2 on femoral bone mineral density as well as in both cortical and cancellous bone was decreased in ERα-C451A mice compared with their wild-type littermates. Histological and biochemical approaches concurred with the results from bone marrow chimeras to demonstrate that ERαMISS signaling affects the osteoblast but not the osteoclast lineage in response to E2. Thus, in contrast to the uterine and endothelial effects of E2 that are specifically mediated by nuclear ERα and ERαMISS effects, respectively, bone protection is dependent on both, underlining the exquisite tissue-specific actions and interactions of membrane and nuclear ERα.