Histochemical examination of systemic administration of eldecalcitol combined with guided bone regeneration for bone defect restoration in rats

The aim of this experiment was to elucidate the histological alterations after systemic administration of eldecalcitol (ELD) combined with guided bone regeneration during the restoration of bone defect healing in rats. The femurs of 8-week-old Wister rats were used to generate bone defect models. The defect was covered with a collagen membrane, and ELD group was administrated with eldecalcitol (50 ng/kg body weight) intragastrically once every other day. Femora were harvested at 1, 2, 4 and 8 weeks post-surgery. Decalcify tissue slices were made and used for histological and immunohistochemical examination. Bone biomarkers of RANKL, OPG and osteocalcin (OCN) were detected by western blot. The results revealed that the system administration of ELD could improve new bone formation demonstrated by the increased bone volume/tissue volume ratio and accelerated mineralization. ELD suppressed osteoclastic bone resorption by reducing the number of osteoclasts, decreasing the expression of cathepsin-K and the ratio of RANKL/OPG at the early stage of bone defect restoration (1 and 2 weeks) and upregulating OCN expression at the later stage of bone defect healing (4 and 8 weeks). These data suggested that systemic administration of eldecalcitol accelerated bone formation and promoted bone maturation by decreasing bone resorption and promoting bone mineralization during bone defect restoration.     

Immunohistochemical detection of an enamel protein-related epitope in rat bone at an early stage of osteogenesis

Monoclonal antibody MI315 was produced against hamster tooth germ homogenate by in vitro immunization. It was found that MI315 reacted with enamel matrix, ameloblasts, and bone matrix at an early stage of osteogenesis. Decalcified tissues of rat femurs and mandibles were examined with MI315 using indirect immunofluorescence. In endochondral ossification of femurs, immunoreactivity was found in bone extracellular matrix (ECM) deposited on the surface of the cartilage core of primary spongiosa, but not in the cartilage core itself. In intramembranous ossification of 0-day-old rat mandibles, intense immunofluorescence was detected in bone ECM and a few young osteocytes, but not in osteoblasts. Immunoreactivity in bone ECM of 2-day-old rats decreased and almost disappeared from bone ECM of 4-day-old rats. Although in nondecalcified sections of 0-day-old rats, negligible immunofluorescence was detected in bone ECM which showed positive staining in decalcified tissues, the immunostaining appeared after decalcification using ethylenediaminetetraacetic acid (EDTA). These results indicate that a substance(s), which had a common epitope with an enamel-derived protein(s), existed in immature bone ECM of both endochondral and intramembranous ossification, and that it might be masked by bone mineral. Monoclonal antibody MI315 is a useful tool to investigate the time- and position-specific changes in osteogenesis and amelogenesis.     

The influence of dietary sodium on bone development in growing rats

The present study investigated the effects of dietary sodium on bone growth in young rats. Five-week-old rats were fed one of three different diets for 60 days: low sodium (NaCl, 0.32 g/kg diet), normal sodium (NaCl, 2.6 g/kg) and high sodium (NaCl, 20 g/kg). The proximal tibial metaphysis (PTM), the fifth lumbar vertebra (LV5) and the middle part of the tibia shaft (TX) were analysed by bone histomorphometry. The expression of three osteogenesis genes, Runx2, osteopontin and osteocalcin, was determined by RT-PCR in bone samples from the skull. In both the PTM and LV5, trabecular area and thickness were increased by the low-sodium diet, while the high-sodium diet decreased trabecular area in LV5. Dynamic data revealed that sodium restriction increased bone formation parameters in the PTM and LV5, but decreased bone resorption in LV5. In TX, endosteal bone formation was enhanced by the low-sodium diet and depressed by the high-sodium diet compared to the normal sodium group. But there were no statistically changes in the cortical bone area of TX. Low-sodium intake significantly enhanced the expression of all three osteogenesis genes compared to the normal sodium group, while high-sodium intake suppressed osteogenic gene expression. Our results suggest that sodium restriction in growing rats promotes bone development by influencing both bone formation and resorption.     

A histochemical localization on Maclura pomifera lectin during osteogenesis

Summary Mandibular condyles of 4-week-old Wistar strain rats and mandibles of ICR strain mice from 14 days gestation stage to 2 days postnatal stage were used to investigate the localization of Maclura pomifera lectin (MPA) during two modes of osteogenesis. During endochondral ossification of the mandibular condyle, MPA was only localized at the peripheral regions of calcified cartilage after the destruction of chondrocyte lacunae. Bone extracellular matrix (ECM) was not reacted with MPA. In intramembranous ossification of mice mandibles, MPA was stained intensively in the early bone ECM. The intensity of the MPA reaction decreased during bone development. In both cases of osteogenesis, chondroclasts and osteoclasts showed the strong affinity to MPA. These results indicated that the time- and position-specific changes within ECM proceeded during osteogenesis and that MPA was the useful probe to detect chondroclasts and osteoclasts.     

BMAL1 regulates balance of osteogenic–osteoclastic function of bone marrow mesenchymal stem cells in type 2 diabetes mellitus through the NF-κB pathway

In bone marrow mesenchymal stem cell (BMSCs), type 2 diabetes mellitus (T2DM) induces metabolic and functional disorders, leading to imbalanced bone resorption and formation and bone loss. Brain and muscle ARNT-like protein 1 (BMAL1) is involved in regulating T2DM-related suppression of BMSCs osteogenesis and bone formation. However, the relationship between BMAL1 and bone remodelling, especially bone resorption in T2DM, is unclear. We investigated the antergic role played by BMAL1 in T2DM-prompted imbalance in BMSCs osteogenic–osteoclastic function. BMAL1 was inhibited and the receptor activator of nuclear factor-κB ligand/osteoprotegerin (RANKL/OPG) ratio was increased in diabetic BMSCs. Inhibitor κB (IκB) expression was decreased, whereas phosphorylated-p65 (p-p65), caspase-3, and p-IκB expression were increased in diabetic BMSCs. BMAL1 overexpression recovered the osteogenesis ability and suppressed osteoclastic induction capability of BMSCs to improve bone metabolism and function, which was partially due to NF-κB pathway activity inhibition. Our results provide evidence about the role of BMAL1 in T2DM-prompted BMSCs differentiation dysfunction, i.e. partially decreasing NF-κB pathway expression. In T2DM, it might be possible to use overexpressed BMAL1 to re-establish the homeostasis of bone metabolism.     

The influence of dietary sodium on bone development in growing rats

The present study investigated the effects of dietary sodium on bone growth in young rats. Five-week-old rats were fed one of three different diets for 60 days: low sodium (NaCl, 0.32 g/kg diet), normal sodium (NaCl, 2.6 g/kg) and high sodium (NaCl, 20 g/kg). The proximal tibial metaphysis (PTM), the fifth lumbar vertebra (LV₅) and the middle part of the tibia shaft (TX) were analysed by bone histomorphometry. The expression of three osteogenesis genes, Runx2, osteopontin and osteocalcin, was determined by RT-PCR in bone samples from the skull. In both the PTM and LV₅, trabecular area and thickness were increased by the low-sodium diet, while the high-sodium diet decreased trabecular area in LV₅. Dynamic data revealed that sodium restriction increased bone formation parameters in the PTM and LV₅, but decreased bone resorption in LV₅. In TX, endosteal bone formation was enhanced by the low-sodium diet and depressed by the high-sodium diet compared to the normal sodium group. But there were no statistically changes in the cortical bone area of TX. Low-sodium intake significantly enhanced the expression of all three osteogenesis genes compared to the normal sodium group, while high-sodium intake suppressed osteogenic gene expression. Our results suggest that sodium restriction in growing rats promotes bone development by influencing both bone formation and resorption.     

Transforming growth factor-beta and the initiation of chondrogenesis and osteogenesis in the rat femur

We have investigated the ability of exogenous transforming growth factor-beta (TGF-beta) to induce osteogenesis and chondrogenesis, critical events in both bone formation and fracture healing. Daily injections of TGF-beta 1 or 2 into the subperiosteal region of newborn rat femurs resulted in localized intramembranous bone formation and chondrogenesis. After cessation of the injections, endochondral ossification occurred, resulting in replacement of cartilage with bone. Gene expression of type II collagen and immunolocalization of types I and II collagen were detected within the TGF-beta-induced cartilage and bone. Moreover, injection of TGF-beta 2 stimulated synthesis of TGF-beta 1 in chondrocytes and osteoblasts within the newly induced bone and cartilage, suggesting positive autoregulation of TGF-beta. TGF-beta 2 was more active in vivo than TGF-beta 1, stimulating formation of a mass that was on the average 375% larger at a comparable dose (p less than 0.001). With either TGF-beta isoform, the dose of the growth factor determined which type of tissue formed, so that the ratio of cartilage formation to intramembranous bone formation decreased as the dose was lowered. For TGF-beta 1, reducing the daily dose from 200 to 20 ng decreased the cartilage/intramembranous bone formation ratio from 3.57 to zero (p less than 0.001). With TGF-beta 2, the same dose change decreased the ratio from 3.71 to 0.28 (p less than 0.001). These data demonstrate that mesenchymal precursor cells in the periosteum are stimulated by TGF-beta to proliferate and differentiate, as occurs in embryologic bone formation and early fracture healing.     

In vivo administration of 1,25-dihydroxyvitamin D3 suppresses the expression of RANKL mRNA in bone of thyroparathyroidectomized rats constantly infused with PTH

It is known that pharmacological or toxic doses of vitamin D induce bone resorption both in vivo and in vitro, whereas physiological doses of the vitamin have a protective effect on bone in vivo. To investigate the discrepancies of the dose-dependent effect of vitamin D on bone resorption, we examined the in vivo effect of 1,25-dihydroxyvitamin D(3) [1,25(OH)(2)D(3)] on the expression of the receptor activator of nuclear factor-kappaB (NF-kappaB) ligand (RANKL) and osteoprotegerin (OPG) mRNAs in bone of thyroparathyroidectomized (TPTX) rats infused with or without parathyroid hormone (PTH). Continuous infusion of 50 ng/h of PTH greatly increased the expression of RANKL mRNA in bone of TPTX rats. Expression of OPG mRNA was not altered by PTH infusion. When graded doses of 1,25(OH)(2)D(3) was daily administered orally for 14 days to normocalcemic TPTX rats constantly infused with PTH, 0.01 and 0.1 microg/kg of 1,25(OH)(2)D(3) inhibited the PTH-induced RANKL mRNA expression, but 0.5 microg/kg of the vitamin did not inhibit it. Regulator of G protein signaling-2 (RGS-2) gene expression was suppressed by 1,25(OH)(2)D(3) dose-dependently, but PTH/PTHrP receptor mRNA expression was not altered. Bone morphometric analyses revealed that 1,25(OH)(2)D(3) suppressed PTH-induced osteoclast number in vivo. These results suggest that pharmacological or toxic doses of 1,25(OH)(2)D(3) stimulate bone resorption by inducing RANKL, but a certain range of physiological doses of the vitamin inhibit PTH-induced bone resorption, the latter mechanism appeared to be mediated, at least in part, by the suppression of the PTH/PTHrP receptor-mediated signaling.     

Vitamin C-deficiency stimulates osteoclastogenesis with an increase in RANK expression

The effects of vitamin C (VC) on osteoclastogenesis were studied in vivo using ascorbate-requiring Osteogenic Disorder Shionogi (ODS) rats and in vitro using bone marrow-derived monocyte/macrophage cells (BMMs). The results confirmed previous findings of increases in the number of osteoclasts and in bone resorption at 2 and 3 weeks, but not 1 week, of VC-deficiency in ODS rats. The mRNA and protein levels of receptor activator nuclear factor kappaB (RANK) ligand and osteoprotegerin, and the mRNA level of macrophage-colony stimulating factor (M-CSF) in the proximal tibia of VC-deficient rats did not differ from those in VC-supplemented control rats. However, the mRNA levels of RANK, c-fos and c-jun were significantly increased at as early as 1 week of VC-deficiency. These results suggested that VC-deficiency stimulated osteoclastogenesis by increasing RANK expression. The osteoclastic differentiation of BMMs was suppressed in the presence of VC. The suppressed osteoclastogenesis was associated with decreased levels of RANK, c-fos and c-jun. The pretreatment of BMMs with VC or PD 98059, a specific inhibitor of extracellular signal regulated kinase (ERK)-activating MEK1, decreased the expression of RANK induced by M-CSF. VC inhibited the M-CSF-induced activation of ERK. These results suggested that VC-deficiency increased osteoclastogenesis by increasing RANK expression mediated through the activation of ERK.     

MURF1 deficiency suppresses unloading-induced effects on osteoblasts and osteoclasts to lead to bone loss

Loss of mechanical stress or unloading causes disuse osteoporosis that leads to fractures and deteriorates body function and affects mortality rate in aged population. This bone loss is due to reduction in osteoblastic bone formation and increase in osteoclastic bone resorption. MuRF1 is a muscle RING finger protein which is involved in muscle wasting and its expression is enhanced in the muscle of mice subjected to disuse condition such as hind limb unloading (HU). However, whether MuRF1 is involved in bone loss due to unloading is not known. We therefore examined the effects of MuRF1 deficiency on unloading-induced bone loss. We conducted hind limb unloading of MuRF1 KO mice and wild-type control mice. Unloading induced about 60% reduction in cancellous bone volume (BV/TV) in WT mice. In contrast, MuRF1 deficiency suppressed unloading-induced cancellous bone loss. The cortical bone mass was also reduced by unloading in WT mice. In contrast, MuRF1 deficiency suppressed this reduction in cortical bone mass. To understand whether the effects of MuRF1 deficiency suppress bone loss is on the side of bone formation or bone resorption, histomorphometry was conducted. Unloading reduced bone osteoblastic formation rate (BFR) in WT. In contrast, MuRF1 deficiency suppressed this reduction. Regarding bone resorption, unloading increased osteoclast number in WT. In contrast, MURF1 deficiency suppressed this osteoclast increase. These data indicated that the ring finger protein, MURF1 is involved in disuse-induced bone loss in both of the two major bone remodeling activities, osteoblastic bone formation and osteoclastic bone resorption.     

Gallium inhibits bone resorption by a direct effect on osteoclasts

Gallium nitrate has been used clinically to treat cancer-related hypercalcemia. It has been suggested that gallium may reduce calcium release from bone by inhibiting bone resorption, but the mechanism(s) involved remain to be elucidated. Therefore, we have examined the effect of gallium on bone resorption in vitro using osteoclasts isolated from neonatal rat long bones cultured on slices of cortical bone. Gallium nitrate (0.01–100 μg/ml) produced a concentration-dependent inhibition of bone resorption. Morphological studies showed that even (100μg/ml) gallium nitrate induced no light microscopical change in osteoclast morphology and did not affect their survival on bone slices. Pretreatment of bone slices with gallium nitrate (100μg/ml for 18 h), followed by extensive washing also inhibited subsequent osteoclastic bone resorption. These results suggest that gallium can be adsorbed onto the calcified surface of bone and inhibit osteoclastic bone resorption.     

Hippuric acid and 3-(3-hydroxyphenyl) propionic acid inhibit murine osteoclastogenesis through RANKL-RANK independent pathway

Nutritional factors influence bone development. Previous studies demonstrated that bone mass significantly increased with suppressed bone resorption in early life of rats fed with AIN-93G semi-purified diets supplemented with 10% whole blueberry (BB) powder for 2 weeks. However, the effects of increased phenolic acids in animal serum due to this diet on bone and bone resorption were unclear. This in vitro and in ex vivo study examined the effects of phenolic hippuric acid (HA) and 3-(3-hydroxyphenyl) propionic acid (3-3-PPA) on osteoclastic cell differentiation and bone resorption. We cultured murine osteoclast (macrophage) cell line, RAW 264.7 cells, and hematopoietic osteoclast progenitor cells (isolated from 4-week-old C57BL6/J mice) with 50 ng/ml of receptor activator of nuclear factor κ-Β ligand (RANKL). Morphologic studies showed decreased osteoclast number with treatment of 2.5% mouse serum from BB diet–fed animals compared with those treated with serum from standard casein diet–fed mice in both RAW 264.7 cell and primary cell cultures. HA and 3-3-PPA, but not 3–4-PPA, had dose-dependent suppressive effects on osteoclastogenesis and osteoclast resorptive activity in Corning osteo-assay plates. Signaling pathway analysis showed that after pretreatment with HA or 3-3-PPA, RANKL-stimulated increase of osteoclastogenic markers, such as nuclear factor of activated T-cells, cytoplasmic 1 and matrix metallopeptidase 9 gene/protein expression were blunted. Inhibitory effects of HA and 3-3-PPA on osteoclastogenesis utilized RANKL/RANK independent mediators. The study revealed that HA and 3-3-PPA significantly inhibited osteoclastogenesis and bone osteoclastic resorptive activity.     

Creating Rigidly Stabilized Fractures for Assessing Intramembranous Ossification,Distraction Osteogenesis,or Healing of Critical Sized Defects

Assessing modes of skeletal repair is essential for developing therapies to be used clinically to treat fractures. Mechanical stability plays a large role in healing of bone injuries. In the worst-case scenario mechanical instability can lead to delayed or non-union in humans. However, motion can also stimulate the healing process. In fractures that have motion cartilage forms to stabilize the fracture bone ends, and this cartilage is gradually replaced by bone through recapitulation of the developmental process of endochondral ossification. In contrast, if a bone fracture is rigidly stabilized bone forms directly via intramembranous ossification. Clinically, both endochondral and intramembranous ossification occur simultaneously. To effectively replicate this process investigators insert a pin into the medullary canal of the fractured bone as described by Bonnarens⁴. This experimental method provides excellent lateral stability while allowing rotational instability to persist. However, our understanding of the mechanisms that regulate these two distinct processes can also be enhanced by experimentally isolating each of these processes. We have developed a stabilization protocol that provides rotational and lateral stabilization. In this model, intramembranous ossification is the only mode of healing that is observed, and healing parameters can be compared among different strains of genetically modified mice ^5-7, after application of bioactive molecules ^8,9, after altering physiological parameters of healing ¹⁰, after modifying the amount or time of stabilization ¹¹, after distraction osteogenesis ¹², after creation of a non-union ¹³, or after creation of a critical sized defect. Here, we illustrate how to apply the modified Ilizarov fixators for studying tibial fracture healing and distraction osteogenesis in mice.     

Hormonal regulation of acid phosphatase release by osteoclasts disaggregated from neonatal rat bone

Osteoclasts disaggregated from neonatal rat long bones and incubated on plastic or glass substrates were found to release a considerable proportion of tartrate-resistant acid phosphatase into culture supernatants. Enzyme release was detectable in the supernatant medium of cultures containing as few as ten cells after 1 hr of incubation and proceeded in a linear manner for the ensuing 6 hr. Calcitonin (1 pg/ml) and cytochalasin B (5 micrograms/ml) inhibited release into the supernatant, suggesting that release represents enzyme secretion. Prostaglandin E1 induced transient inhibition followed by recovery; parathyroid hormone and 1,25(OH)2 vitamin D3 were without influence. Acid phosphatase release in these cultures shows a pattern of hormone responsiveness that coincides with the effects of these hormones on bone resorption by isolated osteoclasts. The extent of acid phosphatase release and its regulation by calciotropic hormones imply a central role for acid hydrolase secretion in osteoclastic bone resorption. The experimental system described in this study may facilitate analysis of the pharmacological hormonal and cellular regulation of osteoclastic function.     

A histochemical localization on Maclura pomifera lectin during osteogenesis

Mandibular condyles of 4-week-old Wistar strain rats and mandibles of ICR strain mice from 14 days gestation stage to 2 days postnatal stage were used to investigate the localization of Maclura pomifera lectin (MPA) during two modes of osteogenesis. During endochondral ossification of the mandibular condyle, MPA was only localized at the peripheral regions of calcified cartilage after the destruction of chondrocyte lacunae. Bone extracellular matrix (ECM) was not reacted with MPA. In intramembranous ossification of mice mandibles, MPA was stained intensively in the early bone ECM. The intensity of the MPA reaction decreased during bone development. In both cases of osteogenesis, chondroclasts and osteoclasts showed the strong affinity to MPA. These results indicated that the time- and position-specific changes within ECM proceeded during osteogenesis and that MPA was the useful probe to detect chondroclasts and osteoclasts.     

G-CSF-induced evacuation of sinusoidal NK cells and the facilitation of liver regeneration in a partial hepatectomy

Since liver regeneration after partial hepatectomy (PHx) is known to improve by pretreatment with recombinant human G-CSF (rhG-CSF), we investigated the mechanism by evaluating the distribution and activity of sinusoidal NK cells. F344 rats were treated with rhG-CSF (250 microg/kg/day) for 5 days before PHx. Pretreatment with rhG-CSF improved the serum ALT levels and DNA biosynthesis of the remnant liver tissues at 20 h after PHx. Notably, the rhG-CSF pretreatment decreased the number of NK cells in the liver determined by immunohistochemistry using anti-NKR-P1A mAb before and at 20 h after PHx with no significant change in the NK activity per cell base, while also increasing the number of NK cells in the peripheral blood detected by flow cytometry. The rhG-CSF induced a pre-PHx downregulation of the IL-12p70 protein levels, while also promoting the post-PHx reduction of the protein levels of IL-12p70 and IFN-gamma. Conversely, rhG-CSF had no effect on the pre-PHx mRNA levels or the PHx-induced upregulation of mRNA levels of TNF-alpha, IL-1beta, IL-6, TGF-beta, IL-10, HGF, and c-Met determined by real-time RT-PCR. These results strongly suggest that rhG-CSF-induced facilitation of liver regeneration is achieved by immunoregulation through the intrahepatic IL-12 downregulation and evacuation of sinusoidal NK cells.     

Dosing-time dependent effect of dexamethasone on bone density in rats

AimsWhile glucocorticoids are widely used to treat patients with various diseases, they often cause adverse effects such as bone fractures. In this study, we investigated whether the decrease in bone density induced by glucocorticoid therapy was ameliorated by optimizing a dosing-time.Main methodsRats were administered with dexamethasone (Dex) orally (1 mg/kg/day) for 6 weeks at a resting or an active period. After the end of the treatment, bone density of femur, biomarkers of bone formation and resorption, and other biomedical variables were measured.Key findingsBone density of femur was significantly decreased by the 6-week treatment with Dex, and the degree of decrease in the 14 HALO (hours after light on) dosing group (an active period) was larger than that in the 2 HALO dosing group (a resting period). Although urinary calcium excretion was accelerated by Dex treatment, secondary hyperparathyroidism was not detected. Histomorphometry analysis showed that Dex suppressed bone resorption, which was larger in the 2 HALO than in the 14 HALO groups. These data indicate that Dex equally suppressed bone formation in the 2 and 14 HALO groups, but inhibited bone resorption more in the 2 HALO than in the 14 HALO groups.SignificanceThis study shows that the decrease in bone density induced by Dex was changed by its dosing-time.     

Stimulation of prostaglandin E2 and bone resorption by recombinant human interleukin 1 alpha in fetal mouse bones

Recombinant human interleukin 1 alpha (rhIL-1 alpha) stimulates prostaglandin E2 and bone resorption in cultured forearm bones of fetal mouse in a dose-dependent manner: the minimal rhIL-1 alpha to elicit a significant bone resorption was 1.6 ng/ml (89 pM). The half maximal concentrations to elicit bone resorption and thymocyte proliferation were 3.3 ng/ml (183 pM) and 0.31 ng/ml (17 pM), respectively. The bone resorbing activity induced by IL-1 was partially inhibited by indomethacin and hydrocortisone, and completely inhibited by anti-IL 1-antibody. There was a good correlation between PGE2 production and bone resorption induced by IL-1 alpha. These results suggest that rhIL-1 alpha stimulates bone resorption at approximately 10 times the concentrations necessary for thymocyte proliferation and that PGE2 produced in the bone is at least in part involved in osteoclastic bone resorption.