Effect of Intermittent Fasting on Glucose Homeostasis and Bone Remodeling in Glucocorticoid-Induced Osteoporosis Rat Model

Background: The present study examined the effect of intermittent fasting (IF) on bone mineral content (BMC) and bone mineral density (BMD) and the markers of bone remodeling in a glucocorticoid-induced osteoporosis (GIO) rat model.Methods: Forty male rats were allocated to 4 groups (N=10 per group): control group of normal rats; control+IF group (normal rats subjected to IF for 16-18 hr daily for 90 days); dexamethasone (DEX) group: (DEX [0.5 mg i.p.] for 90 days); and DEX+IF group (DEX and IF for 90 days). By the end of the experiment, BMD and BMC in the right tibia were measured. Serum levels of the following were measured: glucose; insulin; triglycerides (TGs); total cholesterol; parathyroid hormone (PTH); osteoprotegerin (OPG); receptor activator of nuclear factor-κB (RANK); bone-resorbing cytokines, including bone deoxypyridinoline (DPD), N-terminal telopeptide of collagen type I (NTX-1), and tartrate-resistant acid phosphatase 5b (TRAP-5b); and bone-forming cytokines, including alkaline phosphatase (ALP) and osteocalcin (OC).Results: DEX administration for 90 days resulted in significantly increased serum levels of glucose, insulin, TGs, cholesterol, PTH, OPG, DPD, NTX-1, and TRAP-5b and significantly decreased BMD, BMC, and serum levels of RANK, OC, and ALP (all P<0.05). IF for 90 days significantly improved all these parameters (all P<0.05).Conclusions: IF corrected GIO in rats by inhibiting osteoclastogenesis and PTH secretion and stimulating osteoblast activity.

Effect of Vitamin D-Enriched Gouda-Type Cheese Consumption on Biochemical Markers of Bone Metabolism in Postmenopausal Women in Greece

Considering the role of bone metabolism in understanding the pathogenesis of osteoporosis, the aim of the present study was to examine the effects of vitamin D-enriched cheese on the serum concentrations of the parathyroid hormone (PTH) and certain bone remodeling biomarkers in postmenopausal women in Greece. In a randomised, controlled dietary intervention, 79 postmenopausal women (55–75 years old) were randomly allocated either to a control (CG: n = 39) or an intervention group (IG: n = 40), consuming 60 g of either non-enriched or vitamin D3-enriched Gouda-type cheese (5.7 μg of vitamin D3), respectively, daily and for eight weeks during the winter. The serum concentrations of 25-hydroxy vitamin D (25(OH)D), PTH, bone formation (i.e., osteocalcin, P1NP) and bone resorption (i.e., TRAP-5b) biomarkers were measured. Consumption of the vitamin D-enriched cheese led to higher serum 25(OH)D concentrations of 23.4 ± 6.39 (p = 0.022) and 13.4 ± 1.35 (p < 0.001) nmol/L in vitamin D-insufficient women being at menopause for less and more than 5 years, respectively. In vitamin D-insufficient women that were less than 5 years at menopause, consumption of vitamin D-enriched cheese was also associated with lower serum PTH (Beta −0.63 ± 1.11; p < 0.001) and TRAP-5b (Beta −0.65 ± 0.23; p = 0.004) levels at follow-up, compared with the CG. The present study showed that daily intake of 5.7 μg of vitamin D through enriched cheese increased serum 25(OH)D concentrations, prevented PTH increase and reduced bone resorption in vitamin D-insufficient early postmenopausal women, thus reflecting a food-based solution for reducing osteoporosis risk in this susceptible group.

Remote-controllable bone-targeted delivery of estradiol for the treatment of ovariectomy-induced osteoporosis in rats

Background Osteoporosis (OP) is a systemic skeletal disease marked by bone mass reduction and bone tissue destruction. Hormone replacement therapy is an effective treatment for post-menopausal OP, but estrogen has poor tissue selectivity and severe side effects. Results In this study, we constructed a poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs)-based drug delivery system to co-load 17β estradiol (E2) and iron oxide (Fe3O4) together, modified with alendronate (AL) to achieve bone targeting and realize a magnetically remote-controllable drug release. The NPs were fabricated through the emulsion solvent diffusion method. The particle size was approximately 200 nm while the encapsulation efficiency of E2 was 58.34 ± 9.21%. The NPs were found to be spherical with a homogenous distribution of particle size. The NPs showed good stability, good biocompatibility, high encapsulation ability of E2 and excellent magnetic properties. The NPs could be effectively taken up by Raw 264.7 cells and were effective in enriching drugs in bone tissue. The co-loaded NPs exposed to an external magnetic field ameliorated OVX-induced bone loss through increased BV/TV, decreased Tb.N and Tb.Sp, improved bone strength, increased PINP and OC, and downregulated CTX and TRAP-5b. The haematological index and histopathological analyses displayed the NPs had less side effects on non-skeletal tissues. Conclusions This study presented a remote-controlled release system based on bone-targeted multifunctional NPs and a new potential approach to bone-targeted therapy of OP. Graphic abstract

Investigating the preventive effects of 99Tc-methylenediphosphonate on glucocorticoid-induced osteoporosis rabbit model

Background and Objective: Osteoporosis is a worldwide healthcare challenge. Conventional medications for osteoporosis prevention are not clinically effective or associated with gastrointestinal tract adverse effects. The present study aimed to comparatively investigate the effects of technetium-99 conjugated with methylene diphosphonate (99Tc-MDP) and calcium carbonate and alendronate in prevention and treatment of osteoporosis in glucocorticoid-induced osteoporosis rabbit model through evaluating bone alkaline phosphatase (B-ALP), TRAP-5b levels and histopathological parameters. Method: Forty healthy female New Zealand rabbits were randomly divided into five groups (each n=8), including control group (Control Group), osteoporosis model group (GIO Group), osteoporosis model + 99Tc-MDP group (99Tc-MDP Group), osteoporosis model + alendronate group (Alendronate Group), and osteoporosis model + calcium carbonate group (calcium carbonate Group). Animals in each group were treated with corresponding interventions for 14 weeks. The blood samples were collected at the first and 14th week, and B-ALP and TRAP-5b levels were detected by enzyme-linked immunosorbent assay (ELISA). The rabbits were anesthetized at the 14th week, and pathological cytological observation was performed on both femurs. Results: Age and weights of rabbits in different groups had no statistically significant differences (P>0.05). B-ALP levels in serum of all groups except for Control Group decreased after treatment, but the differences were not statistically significant (P>0.05). TRAP-5b levels in serum of all groups increased after treatment. Specifically, differences in the GIO Group and Calcium carbonate Group were statistically significant (P<0.05), while differences in 99Tc-MDP Group and Alendronate‎ Group were not statistically significant (P<0.05). Pathological sections revealed that Control Group presented normal bone tissue morphology. The bone tissue morphology of the 99Tc-MDP Group and Alendronate Group was similar to Control Group and GIO Group. Moreover, Calcium carbonate Group and GIO Group exhibited similar bone tissue morphology. Conclusions: 99Tc-MDP has preventive effect on the glucocorticoid-induced osteoporotic rabbit model. This osteoporosis preventive effect might be attributed to the capacities of 99Tc-MDP in promoting the osteoblasts generation and inhibiting the generation and reducing the activity of osteoclasts.

Remote-Controllable Bone-Targeted Delivery of Estradiol For the Treatment of Ovariectomy-Induced Osteoporosis in Rats

BackgroundOsteoporosis (OP) is a systemic skeletal disease marked by bone mass reduction and bone tissue destruction. Hormone replacement therapy is an effective treatment for post-menopausal OP, but estrogen has poor tissue selectivity and severe side effects.ResultsIn this study, we constructed a poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs)-based drug delivery system to co-load 17β estradiol (E2) and iron oxide (Fe3O4) together, modified with alendronate (AL) to achieve bone targeting and realize a magnetically remote-controllable drug release. The NPs were fabricated through the emulsion solvent diffusion method. The particle size was approximately 200 nm while the encapsulation efficiency of E2 was 58.34 ± 9.21%. The NPs were found to be spherical with a homogenous distribution of particle size. The NPs showed good stability, good biocompatibility, high encapsulation ability of E2 and excellent magnetic properties. The NPs could be effectively taken up by Raw 264.7 cells and were effective in enriching drugs in bone tissue. The co-loaded NPs exposed to an external magnetic field ameliorated OVX-induced bone loss through increased BV/TV, decreased Tb.N and Tb.Sp, improved bone strength, increased PINP and OC, and downregulated CTX and TRAP-5b. The haematological index and histopathological analyses displayed the NPs had less side effects on non-skeletal tissues.ConclusionsThis study presented a remote-controlled release system based on bone-targeted multifunctional NPs and a new potential approach to bone-targeted therapy of OP.

Effect of infusion irrigation with different irrigating solutions on transient receptor potential vanilloid 5 and intra-articular inflammation in a post-traumatic osteoarthritis rabbit model

Background The incidence of post-traumatic osteoarthritis (PTOA) after anterior cruciate ligament reconstruction (ACLR) is high, but there is still a lack of intra-operative preventive measures. This study aimed to evaluate the effect of different irrigating solutions continuous irrigation on intra-articular inflammation and cartilage degeneration. Methods 66 New Zealand rabbits were randomly divided into normal (N) group, no treatment (NT) group, sodium chloride (NaCl) group, magnesium sulfate (MgSO4) group, and calcium chloride (CaCl2) group. The right knee joint of the experimental group was utilized to construct the model of PTOA, and the left side was utilized as the normal control group. At different time points postoperatively, the blood concentration of hemoglobin and Mg2 + , the synovial fluid concentration of IL-1 β, TNF-α, tartrate-resistant acid phosphatase-5b (TRAP-5b), and Type II Collagen, the gene expression of IL-1 β and MMP-3, and the protein expression of TRPV5 and CaM were detected. Pearson′s linear correlation was employed to identify the possible relationship between the expression of TRAP-5b and the expression of IL-1β, IL-6, TNF-α, and Type II collagen. The hematoxylin and eosin staining (HE), Masson’s trichrome staining, and Alcian blue staining were performed at postoperative 35 days. Osteoarthritis Scoring (OA score) comprised categories including Alcian blue staining, cartilage histology, the cellular density of cartilage, degree of cell disintegration, and formation of chondrocyte cluster were blindly scored by trained researchers at postoperative 35 days. Results There was no statistical difference (P > 0.05) in the hemoglobin concentration between different groups. The concentration of serum Mg2+ in the MgSO4 group was higher than that of the other three groups (P < 0.05) on the same day of operation, then gradually decreased. The expression of IL-1 β, IL-6, and TRAP-5b in synovial fluid increased 5 days after the operation, decreased at 15 days, and then increased again with time in the NT group, NaCl group, and NT group and NaCl group. At 35 days after the operation, the expression of IL-1 β, IL-6, TRAP-5b, and type II collagen in the MgSO4 group were lower than that in the other three groups (except group N) (P < 0.05).The correlation analysis results showed that the TRAP-5b levels correlated positively with IL-1 β, IL-6, TNF-α, and type II collagen concentrations. The histological examination revealed that the surface smoothness of cartilage, the morphology of chondrocytes, the arrangement of collagen fibers, and the density of proteoglycan in the MgSO4 group were better than those in other experimental groups. At 35 days postoperatively, the gene expression of IL-1 β and MMP-3 and the protein expression of CaM and TRPV5 in synovium in the MgSO4 group was lower than that in the NaCl group and CaCl2 group. Conclusion Intra-operative irrigation with magnesium sulfate solution can inhibit the inflammatory factors and the expression of TRPV5, which can also reduce collagen loss and delay cartilage degeneration. Therefore, the use of magnesium sulfate in intra-operative irrigation may be an ideal choice to prevent PTOA.

How to Get Them off?—Assessment of Innovative Techniques for Generation and Detachment of Mature Osteoclasts for Biomaterial Resorption Studies

The fusion process of mononuclear monocytes into multinuclear osteoclasts in vitro is an essential process for the study of osteoclastic resorption of biomaterials. Thereby biomaterials offer many influencing factors such as sample shape, material composition, and surface topography, which can have a decisive influence on the fusion and thus the entire investigation. For the specific investigation of resorption, it can therefore be advantageous to skip the fusion on samples and use mature, predifferentiated osteoclasts directly. However, most conventional detachment methods (cell scraper, accutase), lead to a poor survival rate of osteoclasts or to a loss of function of the cells after their reseeding. In the present study different conventional and novel methods of detachment in combination with different culture surfaces were investigated to obtain optimal osteoclast differentiation, yield, and vitality rates without loss of function. The innovative method—using thermoresponsive surfaces for cultivation and detachment—was found to be best suited. This is in particular due to its ability to maintain osteoclast activity, as proven by TRAP 5b-, CTSK-activity and resorption pits on dentin discs and decellularized osteoblast-derived matrix plates. In conclusion, it is shown, that osteoclasts can be predifferentiated on cell culture dishes and transferred to a reference biomaterial under preservation of osteoclastic resorption activity, providing biomaterial researchers with a novel tool for material characterization.

Impact of Dehydroepiandrosterone (DHEA) on Bone Mineral Density and Bone Mineral Content in a Rat Model of Male Hypogonadism

Objectives: The present study examined the effect DHEA (dehydroepiandrosterone) on bone mineral content (BMC) and bone mineral density (BMD) and biomarkers of bone remodeling in orchidectomized male rats. Material and Methods: A total of 32 male rats were divided equally into four groups (n = 8): (i) control group (C), (ii) control treated with DHEA (Control + DHEA), (iii) orchidectomized (ORCH) group that underwent bilateral orchidectomy and (iv) orchidectomized (ORCH) rats treated with DHEA (ORCH+DHEA). DHEA treatment started 4 weeks after orchidectomy and continued for 12 weeks. After 12 weeks the bone mineral density (BMD) and bone mineral content (BMC) were assayed in the tibia and femur of the right hind limb of each rat. We also measured the serum levels of the bone turnover markers deoxypyridinoline (Dpd), N-telopeptide of type I collagen (NTx), alkaline phosphatase (ALP), tartrate-resistant acid phosphatase 5b (TRAP-5b) and osteocalcin (OC) as well as receptor activator of nuclear factor kappa B (RANK) and osteoprotegerin (OPG). Results: Orchidectomy in rats caused significant reduction in BMD, BMC, serum levels of testosterone, PTH (parathyroid hormone), OPG, OC and ALP with significant rise in serum levels of TRAP-5B, RANK, Dpd and NTx1 (p < 0.05). On the other hand, DHEA therapy for 12 weeks caused significant improvement in all studied parameters except NTx1 (p < 0.05). Conclusions: DHEA corrected hypogonadism-induced osteoporosis in male rats probably via inhibiting osteoclastogenesis, stimulating the activity of osteoblasts and stimulating the secretion of PTH and testosterone.

Cathepsin K regulates localization and secretion of Tartrate-Resistant Acid Phosphatase (TRAP) in TRAP-overexpressing MDA-MB-231 breast cancer cells

Background Tartrate–resistant acid phosphatase (TRAP/ ACP5) belongs to the binuclear metallophosphatase family and is present in two isoforms. The primary translation product is an uncleaved TRAP 5a isoform with low phosphatase activity. TRAP 5a can be post-translationally processed to a cleaved TRAP 5b isoform with high phosphatase activity by e.g. cysteine proteinases, such as Cathepsin K (CtsK). The relevance of the phosphatase activity of TRAP 5b has been demonstrated for proliferation, migration and invasion of cancer cells. TRAP-overexpressing MDA-MB-231 breast cancer cells displayed higher levels of TRAP 5a and efficient processing of TRAP 5a to TRAP 5b protein, but no changes in levels of CtsK when compared to mock-transfected cells. In TRAP-overexpressing cells colocalization of TRAP 5a and proCtsK was augmented, providing a plausible mechanism for generation of TRAP 5b. CtsK expression has been associated with cancer progression and has been pharmacologically targeted in several clinical studies. Results In the current study, CtsK inhibition with MK-0822/Odanacatib did not abrogate the formation of TRAP 5b, but reversibly increased the intracellular levels of a N-terminal fragment of TRAP 5b and reduced secretion of TRAP 5a reversibly. However, MK-0822 treatment neither altered intracellular TRAP activity nor TRAP-dependent cell migration, suggesting involvement of additional proteases in proteolytic processing of TRAP 5a. Notwithstanding, CtsK was shown to be colocalized with TRAP and to be involved in the regulation of secretion of TRAP 5a in a breast cancer cell line, while it still was not essential for processing of TRAP 5a to TRAP 5b isoform. Conclusion In cancer cells multiple proteases are involved in cleaving TRAP 5a to high-activity phosphatase TRAP 5b. However, CtsK-inhibiting treatment was able to reduce secretion TRAP 5a from TRAP-overexpressing cancer cells.

FGF-23 als Knochenmarker bei metabolischen Osteopathien

ZusammenfassungDie Osteoporose ist die führende metabolische Knochenerkrankung des höheren Lebensalters. In den aktuellen Leitlinien des DVO spielen klinische Risikofaktoren ein zunehmend größere Rolle, sowohl im Hinblick auf die Diagnostik als auch auf die Therapie der verschiedenen Formen der Osteoporose. Neben verschiedenen Ko-Morbiditäten weisen diese Patienten häufig eine Einschränkung der Nierenfunktion auf.Die Entdeckung des phosphatsenkenden Hormons FGF-23 und das zunehmende Verständnis zu dessen Rolle im Calcium-Phosphat Stoffwechsel eröffnen neue Einblicke in das Verständnis der komplexen pathophysiologischen Vorgänge metabolischer Osteopathien. FGF-23 wird im Knochen bei ansteigenden Phosphatspiegeln vermehrt gebildet. An der Niere wirkt FGF-23 phosphaturisch. Durch ansteigende FGF-23-Spiegel gelingt es auch bei nachlassender Nierenfunktion, möglichst lange ein Ansteigen des Serumphosphats zu vermeiden. In einer Vielzahl von Studien konnten signifikante Korrelationen zwischen FGF-23-Spiegeln, Phosphatwerten und der kardiovaskulären Mortalität von Patienten mit und ohne Niereninsuffizienz aufgezeigt werden. Studien zur Rolle von FGF-23 im Knochenstoffwechsel liegen bislang nur vereinzelt vor.Ziel der durchgeführten Untersuchung war es, bei Patienten mit Osteoporose und noch weitestgehend erhaltener Nierenfunktion die mögliche diagnostische Bedeutung von FGF-23 im Vergleich zu etablierten Knochenmarkern wie Knochenspezifische alkalische Phosphatase (BAP) und der Tartrat-resistenten sauren Phosphatase Isoform 5b (TRAP-5b) zu betrachten.In einer prospektiven Pilotstudie wurden 55 Patienten mit metabolischen Osteopathien (überwiegend Osteoporose) aus der endokrinologischen Ermächtigungssprechstunde von Prof. Dr. Jehle untersucht. Neben den klinischen Daten und den routinemäßig bestimmten Laborparametern (Calcium, Phosphat, Kreatinin, eGFR, PTH, 25-OH-Vitamin D3, BAP, TRAP-5b) wurde FGF-23 einmalig mit einem spezifischen ELISA gemessen. Zusammenfassend kann gesagt werden, dass die Daten der Pilotstudie zeigen, dass FGF-23 für einzelne Patienten von diagnostischer Relevanz sein kann. Insbesondere erniedrigte Phosphatspiegel können durch die Messung von FGF-23 besser eingeordnet werden. Die weitere Erforschung von FGF-23 und dessen Rolle beim Knochenstoffwechsel scheint vielversprechend.