Effects and Reversibility of Pre- and Post-natal Iron and Omega-3 Fatty Acid Deficiency, Alone and in Combination, on Bone Development in Rats

Both iron and omega-3 (n-3) polyunsaturated fatty acids may play an important role in bone development. The aim of this study was to investigate the effects of pre- and post-natal iron and n-3 fatty acid deficiency (FAD), alone and in combination, on bone development in rats, and to determine whether effects are reversible when a sufficient diet is provided post-weaning. Using a 2×2-factorial design, 56 female Wistar rats were allocated to one of four diets: (1) control, (2) iron deficient (ID), (3) n-3 FAD or (4) ID and n-3 FAD, and were maintained on the respective diets throughout gestation and lactation. At weaning (post-natal day [PND] 21), offspring (n = 24/group; male:female=1:1) were randomly allocated to either continue with their respective diets or to switch to the control diet until PND 42-45. Bone mineral density (BMD) and bone strength were determined using dual X-ray absorptiometry and three-point bending tests, respectively. Pre- and post-natal ID resulted in significantly lower BMD in the spine and bone strength in the left femur. Both ID and n-3 FAD resulted in lower BMD in the right femur, with an additive reduction in the combined ID and n-3 FAD group vs. controls. While negative effects of pre- and post-natal ID alone were reversed in offspring switched to a control diet post-weaning, lower BMD and bone strength persisted in offspring with combined ID and n-3 FAD during the prenatal and early post-natal period. Effects were not sex-specific. These results indicate that ID during early life may negatively influence bone development, with potential additive effects of n-3 FAD. While the effects of ID alone seem reversible, a combined ID and n-3 FAD may result in irreversible deficits in bone development.

Oral administration of melatonin increases plasma calcium and magnesium and improves bone metabolism in aged male mice

We previously reported that the oral administration of melatonin from 4 to 20 months to male mice improved femoral bone strength and bone density during the aging. Additionally, melatonin receptor, MT2, was immunologically detected in both osteoblasts and osteoclasts of the mouse femoral bone. Thus, melatonin can act on both osteoblasts and osteoclasts to maintain bone strength during the aging process. Here, we analyzed plasma calcium (Ca2+), magnesium (Mg2+), and inorganic phosphorus ([PO4]3-) in 20-month-old male mice with or without administration melatonin (15-20 mg/kg/day) in drinking water. We found that plasma Ca2+ and Mg2+ levels in melatonin-treated mice increased significantly as compared with control mice. In [PO4]3-, melatonin administration tended to increase its plasma level, but did not reach statistical significance. The potential association between these divalent ions and metabolism markers of femoral bone was also examined. In the femoral diaphysis, the plasma Ca2+ and Mg2+ concentrations were positively correlated with periosteal and endosteal circumference which were significantly associated with the Strength Strain Index. Therefore, melatonin treatment enlarged femoral diaphysis and enhanced bone strength by increasing mineral depositions. In addition, the plasma melatonin levels were significantly positive correlation with total bone density and critical thickness in the femoral diaphysis. Since we had not observed the primary trabecular bone and osteoclasts in 20-month-old mice previously, it is suggested that plasma Ca2+ and Mg2+ are not elevated due to bone resorption. The increased plasma Ca2+ and Mg2+ by melatonin may originate from the intestinal absorption of these ions since melatonin binds to the vitamin D3 receptor, its activation is known to promote the intestinal absorption of Ca2+.

The Impact of Nano-Crystal Hydroxyapatites on the Regeneration of Bone Defects

Calcium hydroxyapatite is a widely used material for replacing bone defects. However, the effectiveness of nano-crystalline calcium hydroxyapatite produced from eggshells in the replacement of bone defects has not been investigated yet. The study aimed to evaluate the effectiveness of using nano-crystalline calcium hydroxyapatite made from eggshell for the healing of bone defect of the femur in rats. Forty-eight (n=48) rats underwent a surgical procedure to simulate femoral defect. The animals were sub-divided into 4 groups (each with n=12) depending on the methods of bone defect replacement: I control group (CG) (without bone defect replacement); II intervention group (the bone defect was replaced by PRP (PRP); III intervention group (the bone defect was replaced by nano-crystalline hydroxyapatite obtained from eggshell) (HA) and IV interventional group (the bone defect was replaced by a combination of hydroxyapatite and PRP) (HA+PRP). The degree of effectiveness of studied methods was assessed using radiological (on the 14th day), histological (on the 61st day), and biomechanical analysis (on the 61st day). According to radiographic data, the CG group had the lowest level of bone regeneration after 14 days (4.2 ±1.7%). In the HA + PRP group, the level of bone regeneration was 22.1±7.1 %, which was higher in comparison with the rates of consolidation of bone defects in the HA group (20.7± 9.3) (p = 0.023). According to the histo-morphometry data, the rates of bone tissue regeneration in the PRP group (19.8 ±4.2%) were higher in comparison with the CG group (12.7 ± 7.3%), (p>0.05). In the HA+PRP group, bone regeneration rates (48.9±9.4 %) were significantly higher (p=0.001) than in the HA group (35.1±9.8%). According to the results of biomechanical assessment under the maximum stress (121.0722), the maximum bending deformation of the contralateral bone without defect was 0.028746, which was higher than the indicators of the HA+PRP group, where at the maximum stress (90.67979) the bending deformation was 0.024953 (p>0.05). Compared to CG, PRP, and HA, biomechanical bone strength was significantly higher in the HA + PRP group (p≤0.01). At the maximum stress (51.81391), the maximum bending strain in the CG group was 0.03869, which was lower than in the PRP group, where the maximum stress and bending strain were 59.45824 and 0.055171, respectively (p>0.05). However, the bone strength of the HA group was statistically significantly higher compared to the CG and PRP groups (p<0.01). The results demonstrated the effectiveness of the use of nanocrystalline calcium hydroxyapatite obtained from eggshell in the healing of a bone defect. The best results were observed in the group of the combined use of nano-crystalline calcium hydroxyapatite and PRP.

Correlation of ADIPOQ Gene Single Nucleotide Polymorphisms with Bone Strength Index in Middle-Aged and the Elderly of Guangxi Mulam Ethnic Group

Background: Osteoporosis (OP) is a common orthopedic disease in the elderly, and Adiponectin (ADIPOQ) is closely related to bone metabolism. Objective: To determine the relationship between five single nucleotide polymorphism (SNP) loci in the ADIPOQ gene and osteoporosis in middle-aged and elderly Mulam subjects in Hechi, Guangxi. Methods: This case-control study included 297 middle-aged and elderly Mulam subjects with normal bone mass, 49 subjects with reduced bone mass, and 38 subjects with osteoporosis. Five loci (rs266729, rs1063539, rs2241766, rs3774261, rs710445) of the ADIPOQ in the Mulam subjects were genotyped using SNP with multiple-base extension. Results: The bone strength index (SI) of middle-aged and elderly Mulam subjects showed an overall decreasing trend when the subjects were older. Age, muscle mass, and subcutaneous fat content were the main factors influencing the SI in Mulam subjects. The GC genotype of rs266729 and the GA and GG genotypes of rs710445 were significantly correlated with risk of bone loss (p < 0.05). rs2241766 and rs1063539 showed strong LD (D’ > 0.8, r2 > 0.33). rs710445 and rs266729 loci and rs3774261 and rs2241766 loci showed complete LD (D’ = 1). Conclusions: The GC genotype at rs266729 of the ADIPOQ gene, the GA and GG genotypes at rs710445, and the haplotypes CCGAA and GGTAG correlated with osteoporosis (p < 0.05). The allele C of rs1063539, rs266729 and rs710445 may afford protection for osteoporosis. The allele G may be the genetic susceptibility gene for osteoporosis, increasing the risk of osteoporosis.

Exercise Modality Affects Older Adult CT-Derived Muscle and Bone Loss During Caloric Restriction

Caloric restriction (CR) can exacerbate muscle and bone loss. We examined 18-month changes in computed tomography (CT)-derived trunk muscle, and volumetric bone mineral density (vBMD) and finite element-estimated bone strength of the spine and hip in 55 older adults randomized to CR alone or CR plus aerobic (CR+AT) or resistance (CR+RT) training. Trunk muscle area loss trended higher with CR+AT [-16.8 cm2 (95% CI: -26.4,-7.1) vs CR: -6.7 (-12.8,-0.5), CR+RT: -9.0 (-14.5,-3.4)]. Spine vBMD loss trended higher with CR+AT [−0.014 g/cm3 (−0.027,−0.001) vs. CR: −0.005 (−0.022,0.012), CR+RT: −0.004 (−0.019,0.011)], and similarly for vertebral bone strength. Hip vBMD losses trended lower with CR+RT [−0.015 g/cm3 (−0.024,−0.006) vs. CR: −0.027 (−0.036,−0.019), CR+AT: −0.029 (−0.037,−0.020)]. Hip vBMD and trunk muscle losses were positively correlated (r=0.53), and spine vBMD loss tended to increase with trunk muscle loss (r=0.21) and fat infiltration (r=0.17). Collectively, aerobic training was less effective at preserving muscle-bone health during CR.

Osteoprotective Effects in Postmenopausal Osteoporosis Rat Model: Oral Tocotrienol vs. Intraosseous Injection of Tocotrienol-Poly Lactic-Co-Glycolic Acid Combination

Osteoporosis, the most common bone disease, is associated with compromised bone strength and increased risk of fracture. Previous studies have shown that oxidative stress contributes to the progression of osteoporosis. Specifically, for postmenopausal osteoporosis, the reduction in estrogen levels leads to increased oxidative stress in bone remodeling. Tocotrienol, a member of vitamin E that exhibits antioxidant activities, has shown potential as an agent for the treatment of osteoporosis. Most studies on the osteoprotective effects of tocotrienols had used the oral form of tocotrienols, despite their low bioavailability due the lack of transfer proteins and high metabolism in the liver. Several bone studies have utilized tocotrienol combined with a nanocarrier to produce a controlled release of tocotrienol particles into the system. However, the potential of delivering tocotrienol–nanocarrier combination through the intraosseous route has never been explored. In this study, tocotrienol was combined with a nanocarrier, poly lactic-co-glycolic acid (PLGA), and injected intraosseously into the bones of ovariectomized rats to produce targeted and controlled delivery of tocotrienol into the bone microenvironment. This new form of tocotrienol delivery was compared with the conventional oral delivery in terms of their effects on bone parameters. Forty Sprague–Dawley rats were divided into five groups. The first group was sham operated, while other groups were ovariectomized (OVX). Following 2 months, the right tibiae of all the rats were drilled at the metaphysis region to provide access for intraosseous injection. The estrogen group (OVX + ESTO) and tocotrienol group (OVX + TTO) were given daily oral gavages of Premarin (64.5 mg/kg) and annatto-tocotrienol (60 mg/kg), respectively. The locally administered tocotrienol group (OVX + TTL) was given a single intraosseous injection of tocotrienol–PLGA combination. After 8 weeks of treatment, both OVX + TTO and OVX + TTL groups have significantly lower bone markers and higher bone mineral content than the OVX group. In terms of bone microarchitecture, both groups demonstrated significantly higher trabecular separation and connectivity density than the OVX group (p &lt; 0.05). Both groups also showed improvement in bone strength by the significantly higher stress, strain, stiffness, and Young’s modulus parameters. In conclusion, daily oral tocotrienol and one-time intraosseous injection of tocotrienol–PLGA combination were equally effective in offering protection against ovariectomy-induced bone changes.