Bone formation is not coupled to bone resorption in site-specific manner in adult rats

The trabecular bone of the secondary spongiosa of mature rats shows a coupling of bone formation to resorption. It has been clearly shown that in adult man the coupling of formation and resorption involves a site-specific sequence of events, in which bone resorption is normally followed, at the same site, by bone formation. Whether the coupled processes of bone resorption and formation also occur at the same site in the rat is controversial. To elucidate the spatial relationship between bone formation and resorption in the rat, we compared the percentage of crenated and non-crenated cement lines with the percentage of crenated and non-crenated bone surfaces in the proximal tibia of adult rats aged 16 weeks to 2 years. A similar comparison was also made using bone from adult human iliac crest. We found that the trabecular bones of 16-week-old and 7-month-old rats exhibited a low percentage (7–11%) of crenated cement lines, which is opposite to the proportion (88%) we observed in human trabecular bone. In contrast, the surfaces of rat bone trabeculae showed a similar low proportion of crenated surface to human bone (rat 1.1–1.4% vs. 5% in humans). In older (2 years) rats, in which bones have ceased to grow in length, the percentage of cement lines that were crenated increased to 45%. These results imply that the major proportion of bone formation in the trabecular bone of growing rats occurs on non-resorbed surfaces. Thus, although there is substantial evidence that bone formation is coupled to resorption in the rat, such that increased resorption is associated with increased formation, and suppression of resorption suppresses bone formation, bone formation does not necessarily occur on a previously resorbed site. This suggests that in the rat, the processes are not coupled by local release of cytokines or local cell interactions but by some other signal, such as mechanical stimulation. Since site-specificity appears not to be crucial to the coupling of formation to resorption, the greater site-specificity of coupling in man, and in older rats, may be attributable to a more static skeleton, which engenders a closer spatial correlation between bone formation and the resorption that induced it. © 1993 Wiley-Liss, Inc.     

Effect of adrenal steroids on bone resorption in rats.

Rats labeled with strontium-85 (85Sr) were rejected with adrenocortical steroids for 2 wk. The urinary-to-tibial (U/T) 85Sr ratio was used as an index of bone resorption. The glucocorticoids caused an inhibition of skeletal resorption, as judged by the 50% reduction in the U/T ratio, and decreased excretion of hydroxyproline. Thyroidal calcitonin levels were slightly elevated in glucocorticoid-treated animals, suggestive of a possible retardation of calcitonin release. The U/T ratios of thyroparathyroidectomized (TPTX) rats injected with corticosteroids were 50% of control values. The results indicate that glucocorticoids inhibit bone resorption independent of the action of calcitonin. Cortisol treatment increased the tibial density as measured by a radiographic technique. However, bone density was decreased and the U/T ratio increased in steroid-treated rats fed a low-calcium diet. In TPTX cortisol-treated rats, parathyroid extract (PTE) increased the U/T ratio and serum calcium but not to the degree observed in TPTX PTE-injected control animals. These experiments indicate that in rats glucocorticoids inhibit the rate of bone resorption but this effect can be overcome in part by PTE.     

Magnesium deficiency-induced osteoporosis in the rat: uncoupling of bone formation and bone resorption.

Magnesium (Mg) intake has been linked to bone mass and/or rate of bone loss in humans. Experimental Mg deficiency in animal models has resulted in impaired bone growth, osteopenia, and increased skeletal fragility. In order to assess changes in bone and mineral homeostasis that may be responsible, we induced dietary Mg deficiency in adult Simonsen albino rats for 16 weeks. Rats were fed either a low Mg diet (0.002 percent) or a normal control Mg diet (0.063 percent). Blood was obtained at baseline, 4 weeks, 8 weeks, 12 weeks and 16 weeks in both groups for serum Mg, calcium, PTH, and 1.25(OH)2-vitamin D determinations. Femora were harvested at 4 weeks and 16 weeks for mineral analysis and histomorphometry. Serum Mg fell in the Mg depleted group to 0.6 mg/dl (mean) by 16 weeks (controls = 2.0 mg/dl). The serum calcium (Ca) concentration was higher in the Mg depleted animals at 16 weeks, 10.8 mg/dl (controls = 8.9 mg/dl). Serum PTH concentration fell progressively in the Mg deficient rats to 30 pg/ml by week 16 (control = 96 pg/ml). Serum concentration of 1.25(OH)2-vitamin D also fell progressively in the Mg deficient animals by 16 weeks to 14 pg/ml (control = 30 pg/ml). While the percent ash weights of Ca and phosphorus in the femur were not different at any time point, the percent ash weight of Mg progressively fell to 0.54 percent vs control (0.74 percent) by 16 weeks. The percent ash weight of potassium also fell progressively in the Mg deficient group to approximately 30 percent of control by 16 weeks. Histomorphometric analyses showed a significant drop in trabecular bone volume in Mg deficient animals by 16 weeks (percent BV/TV = 13.2 percent vs 17.3 percent in controls). Evaluation of the endosteal bone surface features showed significantly greater bone resorption in the Mg depleted group as reflected in increased number of tartrate-resistant positive osteoclasts/mm bone surface (7.8 vs 4.0 in controls) and an elevated percent of bone surface occupied by osteoclasts (percent OcS/BS = 12.2 percent vs 6.7 percent in controls. This increased resorption occurred in the presence of an inappropriate lowered bone forming surface relative to controls; a decreased number of osteoblasts per mm bone surface (0.23 vs 0.94 in control) and a decrease in percent trabecular surface lined by osteoid (percent OS/BS = 0.41 vs 2.27 percent in controls) were also noted. Our findings demonstrate a Mg-deficiency induced uncoupling of bone formation and bone resorption resulting in a loss of bone mass. While the fall in PTH and/or 1.25(OH)2-D may explain a decrease in osteoblast activity, the mechanism for increased osteoclast activity is unclear. These data suggest that Mg deficiency may be a risk factor for osteoporosis.     

The influence of age on adaptive bone formation and bone resorption

Bone is a tissue with enormous adaptive capacity, balancing resorption and formation processes. It is known that mechanical loading shifts this balance towards an increased formation, leading to enhanced bone mass and mechanical performance. What is not known is how this adaptive response to mechanical loading changes with age. Using dynamic micro-tomography, we show that structural adaptive changes of trabecular bone within the tibia of living mice subjected to two weeks of in vivo cyclic loading are altered by aging. Comparisons of 10, 26 and 78 weeks old animals reveal that the adaptive capacity diminishes. Strikingly, adaptation was asymmetric in that loading increases formation more than it reduces resorption. This asymmetry further shifts the (re)modeling balance towards a net bone loss with age. Loading results in a major increase in the surface area of mineralizing bone. Interestingly, the resorption thickness is independent of loading in trabecular bone in all age groups. This data suggests that during youth, mechanical stimulation induces the recruitment of bone modeling cells whereas in old age, only bone forming cells are affected. These findings provide mechanistic insights into the processes that guide skeletal aging in mice as well as in other mammals.     

Soluble silica inhibits osteoclast formation and bone resorption in vitro

Several studies have suggested that silicon (Si) may be essential for the normal development of connective tissue and the skeleton. Positive effects of Si from the diet as well as from Si-containing biomaterials, such as bioactive glass 45S5 (BG), have been demonstrated. Studies have reported that Si stimulates osteoblast proliferation and differentiation. However, the effects of Si on osteoclasts have not been directly addressed. The purpose of the present in vitro study was to clarify if Si has regulatory effects on osteoclast formation and bone resorption. The effects of BG, BG dissolution extracts and Si containing cell culture medium were investigated in a mouse calvarial bone resorption assay and osteoclast formation assays (mouse bone marrow cultures and RAW264.7 cell cultures). We conclude from our results that Si causes significant inhibition of osteoclast phenotypic gene expressions, osteoclast formation and bone resorption in vitro. In conclusion, the present study suggests that Si has a dual nature in bone metabolism with stimulatory effects on osteoblasts and inhibitory effects on osteoclasts. This suggested property of Si might be interesting to further explore in future biomaterials for treatments of bone defects in patients.     

Cellular and humoral mechanisms of osteoclast formation and bone resorption in Gorham-Stout disease.

Gorham-Stout disease (GSD) is a rare, massively osteolytic condition which is associated with increased vascularity and an increase in osteoclast numbers. To determine the cellular and humoral mechanisms underlying the increase in osteoclast numbers and osteolysis in GSD, this study analysed circulating osteoclast precursor numbers and sensitivity to osteoclastogenic factors in a GSD patient and age/sex-matched controls. Monocytes were cultured with M-CSF (25 ng/ml) and RANKL (30 ng/ml) and osteoclast formation was assessed in terms of the formation of TRAP(+) and VNR(+) multinucleated cells and the extent of lacunar resorption. There was no increase in the proportion of circulating osteoclast precursors in GSD relative to controls, but lacunar resorption was consistently greater in GSD monocyte cultures. Increased osteoclast formation in GSD was noted when monocytes were incubated with IL-1beta (1 ng/ml), IL-6/sIL-6R (100 ng/ml), and TNFalpha (10 ng/ml). An increase in osteoclast differentiation and bone resorption was also noted in control monocyte cultures in the presence of GSD serum. These results indicate that the increase in osteoclast formation in GSD is due not to an increase in the number of circulating osteoclast precursors, but rather to an increase in the sensitivity of these precursors to humoral factors which promote osteoclast formation and bone resorption.     

Ischemic stroke in rats enhances bone resorption in vitro

We hypothesized that the formation and differentialtion of osteoclasts are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow in the early stage of stroke. We randomly divided white female Sprague-Dawley (SD) rats (n = 30) into two groups, stroke (n = 15) and sham group (n = 15). On the 7th day after stroke, after cutting away the epiphyses of the femurs and tibias, diaphyseal channels were flushed using α-minimum essential medium (α-MEM) and bone marrow cells were collected. Bone marrow stem cells, which were extracted from the femur and tibia, were cultured on the 7th day after middle cerebral artery occlusion. We then estimated the ratio of non-adherent cells to total bone marrow cells that included osteoclast precursor cells. After culturing these cells separately, cells that tested positive on the tartrate resistant acid phosphatase (TRAP) were counted and bone resorption was evaluated by using the OAAS™ plate. In comparison to the control group, the stroke group showed a higher increase of non-adherent cells in the hemiplegic side bone marrow. In addition, after the primary culture, the stroke group showed an increased number of TRAP positive cells and a higher degree of bone resorption estimated by OAAS™ plate. As a result, osteoclastogenesis and osteoclast differentiation are accelerated and the potential of bone resorption is increased in the hemiplegic bone marrow and these changes are detected as early as within the first week after middle cerebral artery occlusion in SD rats.     

Function of matrix IGF-1 in coupling bone resorption and formation

Balancing bone resorption and formation is the quintessential component for the prevention of osteoporosis. Signals that determine the recruitment, replication, differentiation, function, and apoptosis of osteoblasts and osteoclasts direct bone remodeling and determine whether bone tissue is gained, lost, or balanced. Therefore, understanding the signaling pathways involved in the coupling process will help develop further targets for osteoporosis therapy, by blocking bone resorption or enhancing bone formation in a space- and time-dependent manner. Insulin-like growth factor type 1 (IGF-1) has long been known to play a role in bone strength. It is one of the most abundant substances in the bone matrix, circulates systemically and is secreted locally, and has a direct relationship with bone mineral density. Recent data has helped further our understanding of the direct role of IGF-1 signaling in coupling bone remodeling which will be discussed in this review. The bone marrow microenvironment plays a critical role in the fate of mesenchymal stem cells and hematopoietic stem cells and thus how IGF-1 interacts with other factors in the microenvironment are equally important. While previous clinical trials with IGF-1 administration have been unsuccessful at enhancing bone formation, advances in basic science studies have provided insight into further mechanisms that should be considered for future trials. Additional basic science studies dissecting the regulation and the function of matrix IGF-1 in modeling and remodeling will continue to provide further insight for future directions for anabolic therapies for osteoporosis.     

Acidification of the osteoclastic resorption compartment provides insight into the coupling of bone formation to bone resorption

Patients with defective osteoclastic acidification have increased numbers of osteoclasts, with decreased resorption, but bone formation that remains unchanged. We demonstrate that osteoclast survival is increased when acidification is impaired, and that impairment of acidification results in inhibition of bone resorption without inhibition of bone formation. We investigated the role of acidification in human osteoclastic resorption and life span in vitro using inhibitors of chloride channels (NS5818/NS3696), the proton pump (bafilomycin) and cathepsin K. We found that bafilomycin and NS5818 dose dependently inhibited acidification of the osteoclastic resorption compartment and bone resorption. Inhibition of bone resorption by inhibition of acidification, but not cathepsin K inhibition, augmented osteoclast survival, which resulted in a 150 to 300% increase in osteoclasts compared to controls. We investigated the effect of inhibition of osteoclastic acidification in vivo by using the rat ovariectomy model with twice daily oral dosing of NS3696 at 50 mg/kg for 6 weeks. We observed a 60% decrease in resorption (DPYR), increased tartrate-resistant acid phosphatase levels, and no effect on bone formation evaluated by osteocalcin. We speculate that attenuated acidification inhibits dissolution of the inorganic phase of bone and results in an increased number of nonresorbing osteoclasts that are responsible for the coupling to normal bone formation. Thus, we suggest that acidification is essential for normal bone remodeling and that attenuated acidification leads to uncoupling with decreased bone resorption and unaffected bone formation.     

Maternal diet affects feeding behaviour of self-selecting weanling rats

Weanling rats have been shown to regulate protein intake when provided the opportunity. The objective of these studies was to determine the influence of protein concentration in the maternal diet on protein selection of the offspring. Dams were fed single diets containing 10, 20, 30, or 40% casein throughout gestation and lactation. Pups from all groups were then allowed to select from diets containing 10 and 60% casein for two weeks. Protein selection in the weanling rat was found to correlate with protein concentration in the maternal diet expressed either as g/100g BW (r=0.48, p<0.05) or protein concentration (r=0.65, p<0.01). In order to differentiate between a gestational and lactational effect a cross-over study was performed. Pups from dams fed 10% casein during gestation and/or lactation selected 17% protein concentration, lower than the 25.1% selected by pups from dams receiving 40% casein through gestation and lactation. Therefore, feeding behaviour, as indicated by protein selection, is influenced by maternal diet during both gestation and lactation. The use of the self-selection paradigm has been proposed as a behavioural model for the investigation of the functional significance of maternal nutrition.     

Bacterial porins stimulate bone resorption.

Porins are abundant outer membrane proteins of gram-negative bacteria involved in transport of low-molecular-mass molecules. During the past decade, porins from a number of bacteria have also been shown to have proinflammatory activities including inducing the synthesis of proinflammatory mediators (cytokines, platelet-activating factor, and nitric oxide) in cultured cells and inducing inflammation in vivo. With this range of actions, it was possible that porins could also interact with bone cells to cause aberrant bone remodeling and that this could contribute to the bone destruction seen in gram-negative bone infections. By using purified preparations of Salmonella typhimurium and Pseudomonas aeruginosa porins, in the presence of polymyxin B, it was possible to induce concentration-dependent loss of calcium from cultured murine calvaria at porin concentrations in the range of 1 to 10 nM. The mechanism of action of the porins was determined by the inclusion of inhibitors of cyclooxygenase or inflammatory cytokines in the culture media. The bone-resorbing activity of both porins was not inhibited by the cyclooxygenase inhibitor indomethacin or by neutralizing the activity of tumor necrosis factor. Indeed, relatively high concentrations of these agents produced an unexpected increase in the bone resorption induced by the porins. In contrast, porin-induced bone resorption could be inhibited by relatively high concentrations of the natural inhibitor of interleukin-1 (IL-1 receptor antagonist). It appears that these porins stimulate bone resorption by a mechanism distinct from that of lipopolysaccharide, and the possibility therefore exists that porins play a role in bone destruction in gram-negative bacterial infections of bone.     

Salicylic acid-derived poly(anhydride-esters) inhibit bone resorption and formation in vivo

The objective of this study was to investigate the effects of a novel polymer that biodegrades into salicylic acid (SA) on the healing of critical sized long bone defects. Microspheres of the homopolymer, or a copolymer containing 50% less of the SA, were packed into 5-mm mid-diaphyseal defects in rat femurs. Control animals received collagen sponge implants. After 4 and 8 weeks of implantation, bone healing was evaluated using microradiography and quantitative histomorphometry. Four weeks postsurgery, significantly less new bone was formed in both of the polymer groups (p<0.038). Reduced bone loss was also noted with the polymers at this time, although it was not statistically significant. However, at 8 weeks postsurgery, a statistically significant reduction in bone loss was observed in both of the polymer groups compared with controls (p<0.0072). Both polymers seemed to elicit identical tissue responses because there were no differences detected between the homopolymer and copolymer materials at either time point. These results indicate that locally released SA can significantly reduce both bone loss and bone formation in this animal model.     

Induction of nonspecific tolerance to endotoxins reduces the alveolar bone resorption in ligature-treated rats.

Previous experimental data from various laboratories indicate that endotoxin of gram-negative oral microorganisms might be one of the most important bacterial products involved in bone resorption during periodontitis. Immunologically nonspecific tolerance to endotoxins in rats was induced by repeated application of Serratia marcescens trichloroacetic acid-extracted endotoxin. Silk ligature was placed on the second maxillary molar of the endotoxin-tolerant rats as well as of control rats in which tolerance to endotoxin had not been induced. The animals were sacrificed 8 days later. The rats showed no specific immune response to the tolerance-inducing endotoxin as measured by passive hemagglutination and by the lymphoblast assays, but we found that bone resorption was significantly reduced in the endotoxin-tolerant rats as compared with ligature-treated animals in which tolerance to endotoxin had not been induced.     

仙灵骨葆对骨质疏松大鼠骨量及骨髓基质干细胞成骨分化的影响

背景：双侧卵巢切除可造成大鼠骨量丢失,仙灵骨葆作为传统中药具有一定的促进骨形成作用。 目的：观察仙灵骨葆体内给药对骨质疏松大鼠骨量及骨髓基质干细胞成骨分化能力以及相关因子表达的影响。 方法：3月龄雌性SD大鼠24只随机数字表法均分为3组,卵巢切除组和仙灵骨葆组行卵巢切除,造模6周后仙灵骨葆组给予仙灵骨葆250 mg/(kg•d),干预8周;正常对照组不予干预。 结果与结论：卵巢切除组L2-L4椎体骨密度显著低于其他两组,仙灵骨葆组仍显著低于正常对照组(P < 0.05);卵巢切除组血清骨钙素、骨髓基质干细胞的骨形态发生蛋白2、骨钙素mRNA水平均低于其他两组(P < 0.05)。细胞外基质矿化能力亦明显低于正常对照组和仙灵骨葆组。提示大鼠去势14周后骨量丢失显著,仙灵骨葆可部分阻止其骨量丢失,其作用机制可能与促进大鼠骨髓基质干细胞的成骨分化有关。     

Local effects of impaired mechanical properties of collagen on bone formation and resorption

Summary To study the relationship between the mechanical properties of collagen and the bone turnover, 2-week-old Balb/C mice were fed on a diet containing 0.25% Beta aminopropionitrile (B-APN), a potent inhibitor of collagen crosslink formation, for 3 weeks. Mandibular incisor socket was selected for the analysis of bone formation and resorption parameters. Plastic embedded sections stained with toludine blue and cut at 4 µm were used to analyze the average area of bone lamellae, bone-forming surface, and the number of osteoblasts/mm of forming surface. Similar sections were used to localize acid phosphatase on resorbing surfaces and within the osteoclasts, while bone alkaline phosphatase was determined by a colorimetric method. Morphometric analyses showed that the area of newly formed bone lamellae, total forming surface, number of osteoblasts and the Alk. Pase activity were significantly lower in B-APN-fed mice as compared to the controls. There was a concomitant smaller, but significant, reduction in total resorption surface, active resorption surface and the number of osteoclasts. These results suggest that the regulation of bone formation and resorption at this site, which is independent of systemic regulation, is influenced by the mechanical properties of the collagenous matrix, which in turn may have a significant effect on the existing pool of bone-forming cells, but may not influence the recruitment of new cells.     

The effect of simvastatin on bone formation and ceramic resorption in a peri-implant defect model

Experimental use of statins as stimulators of bone formation suggests they may have widespread applicability in the field of orthopaedics. With their combined effects on osteoblasts and osteoclasts, statins have the potential to enhance resorption of synthetic materials and improve bone ingrowth. In this study, the effect of oral and local administration of simvastatin to a beta tricalcium phosphate (betaTCP)-filled defect around an implant was compared with recombinant human bone morphogenetic protein 2 (rhBMP2). On hundred and sixty-two Sprague-Dawley rats were assigned to treatment groups: local application of 0.1, 0.9 or 1.7 mg of simvastatin, oral simvastatin at 5, 10 or 50 mg kg(-1) day(-1) for 20 days, local delivery of 1 or 10 microg of rhBMP2, or control. At 6 weeks rhBMP2 increased serum tartrate-resistant acid phosphatase 5b levels and reduced betaTCP area fraction, particle size and number compared with control, suggesting increased osteoclast activity. There was reduced stiffness and increased mechanical strength with this treatment. Local simvastatin resulted in a decreased mineral apposition rate at 6 weeks and increased fibrous area fraction, betaTCP area fraction, particle size and number at 26 weeks. Oral simvastatin had no effect compared with control. Local application of rhBMP2 increased resorption and improved mechanical strength whereas simvastatin was detrimental to healing. Oral simvastatin was ineffective at promoting either ceramic resorption or bone formation. The effect of statins on the repair of bone defects with graft substitute materials is influenced by its bioavailability. Thus, further studies on the optimal delivery system are needed.     

Porphyromonas gingivalis fimbriae stimulate bone resorption in vitro.

Our previous study demonstrated that Porphyromonas gingivalis fimbriae induce the expression of interleukin-1, a potent bone-resorbing cytokine, in macrophages. This demonstration suggested to use the possibility that the fimbriae may stimulate bone resorption via the generation of an inflammatory cytokine(s). The present study was performed to test this suggestion. The bone-resorbing activity was evaluated by measuring the area of resorption lacunae on bone slices incubated with calvarial bone cells taken from 14-day-old mouse embryos. Fimbriae at 0.5 micrograms of protein per ml stimulated the bone-resorbing activity significantly, and the effect was dose and treatment time dependent. Since it is well known that interleukin-1 and granulocyte macrophage colony-stimulating factor induce differentiation of osteoclast lineage cells, we examined the involvement of these cytokines in fimbria-stimulated bone resorption. Fimbria-stimulated bone resorption was abolished significantly by antisera against both cytokines. We observed by Northern (RNA) blot assay that both cytokine genes were markedly expressed in the fimbria-treated calvarial bone cells. Our present data demonstrate that P. gingivalis fimbriae stimulate bone resorption in vitro.