Rat Trabecular Bone Research Articles

SCIMP: A Novel Targeted Gene for Postmenopausal Osteoporosis Progression.

The literature suggests that not all postmenopausal women suffer from osteoporosis, and the occurrence of postmenopausal osteoporosis is closely related to the genetic susceptibility of genes in the population and the cellular pathways of related genes. To systematically understand the functions of SCIMP gene for osteoporosis, both in vitro and in vivo experiments were analyzed in depth in this integrated study. The significantly differentially expressed genes of postmenopausal osteoporosis (PMOP) patients from GEO database were selected. Meanwhile, the primary target gene was also confirmed in clinically recruited individuals using ELISA method; 50 postmenopausal osteoporosis patients with a T-score of -2.5 were randomly enrolled; postmenopausal women with a T-score > -2.5 were included in the non-osteoporotic group (including osteopenia and normal bone mineral density). The associated processes and signaling pathways were deeply investigated with GO and KEGG enrichment analysis. The downstream signaling factors including Erk-1/2, Akt, and IkB-related signaling pathways for the potential gene were evaluated using MG-63 cell line; the MTT, CCK-8, and flow cytometry assays were performed to exam MG-63 cell viability, proliferation, as well as apoptosis, respectively, under different treatments. Based on the differentially expressed gene analysis for GEO database, PMOP patients displayed 845 differentially expressed genes, including 709 down-regulated and 136 up-regulated ones. Ten genes including SCIMP were significantly differentially expressed (at least three-fold difference). SCIMP was the most markedly decreased in PMOP patients' specimens. Using clinical recruited individuals, the concentration of SCIMP was 96.6 ± 20.8 ng/μL in the PMOP group compared with 168.8 ± 23.5 ng/μL in the control group (p < 0.05). At the same time, the osteoclast differentiation signaling pathway was significantly up-regulated while hedgehogs as well as other signaling pathways were down-regulated based on the KEGG analysis. The phosphorylation level of Akt was markedly blocked in si-SCIMP treatment. Up-regulation of SCIMP increased cell proliferation, inhibited cell apoptosis, and enhanced cell viability in MG-63 cells, which was markedly rescued by AKT phosphorylation inhibitor. Finally, in vivo experiments also confirmed that the upregulation of SCIMP enhanced the structural parameters of rat trabecular bone and the osteogenic activity of bone tissue. SCIMP plays a critical role in the pathogenesis of postmenopausal osteoporosis in women. SCIMP influences osteoclasts function through an akt-dependent molecular pathway, and subsequently influences the equilibrium process of bone metabolism. This provides a new insight into the pathogenesis of postmenopausal osteoporosis as well as the clinical treatment of osteoporosis.

Quantification of skeletal growth, modeling, and remodeling by in vivo micro computed tomography

In this study we established an image analysis scheme for the investigation of cortical and trabecular bone development during skeletal growth and tested this concept on in vivo μCT images of rats. To evaluate its efficacy, we applied the technique to young (1-month-old) and adult (3-month-old) rat tibiae with vehicle (Veh) or intermittent parathyroid hormone (PTH) treatment. By overlaying 2 sequential scans based on their distinct trabecular microarchitecture, we calculated the linear growth rate of young rats to be 0.31mm/day at the proximal tibia. Due to rapid growth (3.7mm in 12days), the scanned bone region at day 12 had no overlap with the bone tissue scanned at day 0. Instead, the imaged bone region at day 12 represented newly generated bone tissue from the growth plate. The new bone of the PTH-treated rats had significantly greater trabecular bone volume fraction, number, and thickness than those of the Veh-treated rats, indicating PTH's anabolic effect on bone modeling. In contrast, the effect of PTH on adult rat trabecular bone was found to be caused by PTH's anabolic effect on bone remodeling. The cortical bone at the proximal tibia of young rats also thickened more in the PTH group (23%) than the Veh group (14%). This was primarily driven by endosteal bone formation and coalescence of trabecular bone into the cortex. This process can be visualized by aligning the local bone structural changes using image registration. As a result, the cortex after PTH treatment was 31% less porous, and had a 22% greater polar moment of inertia compared to the Veh group. Lastly, we monitored the longitudinal bone growth in adult rats by measuring the distance of bone flow away from the proximal tibial growth plate from 3months to 19months of age and discovered a total of 3.5mm growth in 16months. It was demonstrated that this image analysis scheme can efficiently evaluate bone growth, bone modeling, and bone remodeling, and is ready to be translated into a clinical imaging platform.

Membrane-bound carbonic anhydrases in osteoclasts

Osteoclasts are multinucleated bone-resorbing cells that use multiple pH regulation mechanisms to create an acidic pH in the resorption lacuna. Carbonic anhydrase II and vacuolar H(+)-ATPases produce and transport protons, while chloride channels provide a Cl(-) flux into the resorption site. These activities are required for inorganic matrix dissolution that precedes enzymatic removal of organic bone matrix. In other cell types it has become evident that carbonic anhydrase isoenzymes interact with AE proteins to form transport metabolons that regulate intracellular pH. Membrane-bound carbonic anhydrase isoenzymes may also compensate for the lack of cytoplasmic carbonic anhydrase II. Therefore, our goal was to explore the expression of membrane-bound carbonic anhydrase (CA) isoenzymes CA IV, CA IX, CA XII and CA XIV in bone-resorbing osteoclasts. Immunohistochemistry and confocal microscopy showed expression of CA IV, CA XII and CA XIV in cultured rat and human osteoclasts. To confirm these results, RT-PCR was used. Immunohistochemistry revealed distinct staining patterns for CA IV, CA XII and CA XIV in rat trabecular bone specimens. A plasma membrane staining was observed in bone lining cells with the CA XII antibody while osteoclast plasma membranes were stained with CA IV and CA XIV antibodies. Confocal microscopy of cultured human osteoclasts showed a punctated intracellular CA IV staining and a perinuclear CA XIV staining while no CA IX or CA XII staining was observed. To evaluate the physiological role of membrane-bound CAs in osteoclasts, we used PCS, a novel membrane-impermeable CA inhibitor. Increased osteoclast number and bone resorption activity was observed in rat osteoclast cultures exposed to a low concentration of PCS while higher concentrations affected cell survival. PCS treatment also disturbed intracellular acidification in osteoclasts, as determined by live cell microscopy. In conclusion, our data shows that membrane-bound carbonic anhydrase isoenzymes CA IV and CA XIV are expressed both at mRNA and protein levels in osteoclasts in vivo and in vitro. In addition, the inhibitor experiments provide novel evidence to support the hypothesis that intracellular pH regulation in osteoclasts may indeed involve transport metabolons.

Effects of in vitro bone formation on the mechanical properties of a trabeculated hydroxyapatite bone substitute

This study was designed to test the hypothesis that the mechanical properties of a trabecular bone substitute can be enhanced through in vitro tissue formation. Our specific objectives were to (1) determine the effects of in vitro marrow stromal cell-mediated tissue deposition upon a trabeculated hydroxyapatite scaffold on the strength and toughness of the resulting bone substitute; and (2) identify and characterize regions of newly deposited matrix and mineral. This work provides a basis for future investigations aimed at transforming a brittle hydroxyapatite scaffold into an osteoinductive, biomechanically functional implant through in vitro bone deposition. As hypothesized, the mechanical properties of the trabecular bone substitutes were significantly enhanced by in vitro tissue formation. As a result of cell seeding and a 5 week culture protocol, mean strength increased by 85% (p = 0.008) and energy to fracture increased by 130% (p = 0.003). Accompanying the enhancement of mechanical properties was the deposition of significant amounts of bone matrix and mineral. Fluorescence imaging, scanning electron microscopy, electron probe microanalysis, and nanoindentation confirmed the presence of bonelike mineral with Ca/P ratio, modulus, and hardness similar to that within human and rat trabecular bone tissue. This new mineralization was found to exist within a newly deposited parallel-fibered matrix both encasing and bridging between scaffold trabeculae. Taken as a whole, our results establish the feasibility of the production of an osteoinductive hydroxyapatite-based trabecular bone substitute with mechanical properties enhanced through in vitro bone deposition.

High-resolution micro-CT evaluation of mid- to long-term effects of estrogen deficiency on rat trabecular bone 1

Rationale and Objectives. Previous studies have shown dramatic short-term bone loss following ovariectomy. The purpose of this study was to evaluate mid- to long-term effects of estrogen deficiency on microarchitecture of tibial trabecular bone using a high-resolution microcomputed tomography (Micro-CT). Materials and Methods. Twenty-eight female rats were divided into two equal groups: ovariectomized (OVX, n = 14) and sham-operated (SOVX, n = 14), which in turn were divided into two equal subgroups (euthanized either 6 or 16 weeks after surgery). The left tibia of each animal was scanned with a high-resolution micro-CT (Skyscan 1072 micro-CT system; SkyScan, Aartselaar, Belgium). The Micro-CT system used an X-ray CCD-camera with a cooled 1024 × 1024-pixel 12-bit sensor. Results. Ovariectomy significantly decreased the ratio of bone volume:tissue volume (−42% at 6 weeks and −69% at 16 weeks) and trabecular thickness (−13% at 6 weeks and −30% at 16 weeks), while significantly increasing trabecular separation (+73% at 6 weeks and +100% at 16 weeks) and structure model index (+15% at 6 weeks and +48% at 16 weeks), when compared with SOVX, from 6 to 16 weeks. Conclusion. Based on the experimental results, there are alterations of trabecular microarcihtecture in the tibia following mid- and long-term estrogen deficiency. More rod-shaped trabeculae are formed with increasing duration of estrogen deficiency. The high-resolution micro-CT imaging system is useful to evaluate the mid- to long-term trabecular changes seen with estrogen deficiency or osteoporosis.

Pre-osteoblastic proliferation assessed with BrdU in undecalcified, Epon-embedded adult rat trabecular bone.

We evaluated bromodeoxyuridine (BrdU) immunohistochemistry in undecalcified adult rat tibiae to study cell kinetics in various bone compartments: primary and secondary spongiosae, periosteum, and bone marrow. Several regimens of BrdU administration were tested (i.p. injections and osmotic minipumps). We compared LR White resin, methylmethacrylate, and Epon-araldite embedding, microwave irradiation for antigen retrieval, several concentrations of sodium ethoxide for deplastification, and various DNA denaturation procedures. Paraffin-embedded decalcified tibiae and Epon-embedded bowel were used as positive controls. The best results were obtained in rats labeled with 40 mg of BrdU for 72 hr using osmotic minipumps. The procedure using a Microprobe system in Eponembedded bone tissue with a sodium ethoxide concentration of 50% for two intervals of 20 min provided the best staining quality and tissue preservation. Labeled pre-osteoblastic cells and bone marrow cells could be counted. Epon embedding allowed preservation of tetracycline double labeling performed 1 to 5 days before sacrifice. The number of labeled pre-osteoblastic cells was correlated with the double-labeled surface area measured histomorphometrically.

17 beta-Estradiol and tamoxifen prevent the over-glycosylation of rat trabecular bone collagen induced by ovariectomy.

We have recently demonstrated that ovariectomy in the rat causes over-glycosylation of collagen which is restricted to trabecular bone. In order to obtain further evidence, we studied whether estrogen or tamoxifen treatment prevented over-glycosylation of trabecular bone collagen. Forty one-hundred-day-old female rats were subjected to ovariectomy (n = 30) or sham-operation (n = 10). Starting the day of the operation, sham-operated rats were treated with vehicle, while ovariectomized rats were divided into three groups and treated with vehicle (n = 10), estrogen (n = 10) or tamoxifen (n = 10). Five rats from each group were sacrificed at 115 and 145 days of age. Femurs and tibiae were separated into cortical and trabecular bone, demineralized, hydrolyzed and analyzed by HPLC for hydroxylysine glycoside and hydroxyproline content. Hydroxylysine glycoside content was expressed as a molar ratio with hydroxyproline. The results can be summarized as follows: 1) cortical bone collagen glycosylation did not vary among the different groups; 2) over-glycosylation of trabecular bone collagen observed in the ovariectomized rats was prevented by the administration of either 17 beta-estradiol or tamoxifen. These data demonstrated that estrogens affect glycosylation of trabecular bone collagen.

P39. The influence of parathyroidectomy (PTX) and ovariectomy (OVX) upon matrix metalloproteinases within rat trabecular bone

P39. The influence of parathyroidectomy (PTX) and ovariectomy (OVX) upon matrix metalloproteinases within rat trabecular bone

Opposite effects of insulin-like growth factor-I on the formation of trabecular and cortical bone in adult female rats.

We have previously found that in intact adult female rats, 17 beta-estradiol (E2) stimulates trabecular bone formation, which can be detected by triple fluorochrome labeling within a few days of commencing E2 treatment. We have also observed that E2 stimulates trabecular bone formation in ovariectomised (Ovx) rats given 3-amino-1-hydroxypropylidene bisphosphonate (AHPrBP). This anabolic action of E2 is presumably mediated through the local release of growth factors, of which insulin-like growth factor-I (IGF-I) is a strong contender. To assess the role of IGF-I as mediator of the anabolic action of E2 on rat trabecular bone, we examined the effect of daily sc administration of IGF-I (200 micrograms/kg) for 17 days on trabecular bone formation at the proximal tibial metaphysis in experiments using the above two models. Intact animals received fluorochrome labels on days 1, 8, and 15 of IGF-I treatment, and Ovx animals received AHPrBP (0.3 mg/kg) on days 1, 8, and 15 and fluorochrome labels on days 8 and 15. Trabecular surfaces covered by first and second labels only (termed arrested surfaces), second and third labels only (induction surfaces), and all three labels (persisting surfaces) were assessed in intact animals; double labeled surfaces were assessed in Ovx animals. We found that IGF-I increased the longitudinal growth rate in both intact rats and Ovx animals given AHPrBP. However, to our surprise, we found that IGF-I inhibited trabecular bone formation in intact animals, reducing both arrested and persisting trabecular surfaces by approximately 50%. This was associated with a doubling of periosteal bone formation, as measured at the tibial diaphysis. In Ovx animals given AHPrBP, the AHPrBP markedly inhibited trabecular bone formation compared with that in Ovx controls, and IGF-I was without effect. We conclude that, in contrast to E2, IGF-I acts to increase the length and circumference of long bones and, perhaps through reactive mechanisms such as mechanical compensation, reduces rather than stimulates trabecular bone formation.

The anabolic action of 17β-estradiol (E 2) on rat trabecular bone is suppressed by (3-amino-1-hydroxypropylidene)-1-bisphosphonate (AHPrBP)

We have previously found that high doses of 17 beta-estradiol (E2), similar to those seen in late pregnancy, stimulate bone formation in adult rats. In this communication we tested the effects of a combination of E2 and (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) on bone formation and bone volume in rat bone. E2 (4 mg/kg/day subcutaneously for 17 days) stimulated the bone formation rate to 6 times that of control rats. This was reduced by a single administration of AHPrBP (0.3 mg/kg subcutaneously) to 3 times control levels, and by similar daily injections of AHPrBP to levels not significantly different from those of untreated rats. Suppression of bone formation was effected predominantly through a reduction in the percentage of double-labelled surfaces, consistent with reduced osteoblast recruitment. We found only relatively minor effects of AHPrBP on the mineral apposition rate, suggesting that AHPrBP affected osteoblast function less than osteoblast recruitment. Suppression of histodynamic parameters of bone formation by AHPrBP was associated with suppression of the increase in bone volume otherwise induced by E2. The suppression by AHPrBP of the effect of E2 on bone formation contrasted with its lack of effect on other target tissues for E2, since AHPrBP did not affect the E2-induced changes in longitudinal bone growth or uterine weight. These results suggest that AHPrBP inhibits the anabolic effect of estrogen on rat trabecular bone.

A difference in the enzyme contents of resorption lacunae and secondary lysosomes of osteoclasts.

The distributions of tartrate sensitive lysosomal acid phosphatase (LAP) and cathepsin L in osteoclasts and the effect of parathyroid hormone (PTH) stimulation on them were investigated by using the protein A-gold method on ultracryosections of rat trabecular bone. LAP was located in association with the ruffled border membrane, in the resorption lacuna, on the mineral phase surrounding the lacuna, and in some primary lysosomes. After PTH treatment, the extracellular and ruffled border membrane associated LAP apparently increased. Heavy gold labelling for cathepsin L was confined exclusively to secondary lysosomes. No labelling was seen in the extracellular resorption lacuna or at the ruffled border. Acceleration of bone resorption by PTH-treatment did not change detectably the distribution or intensity of labelling. This study shows that the enzyme contents of secondary lysosomes and resorption lacunae are different and suggests that LAP is directly involved in extracellular bone degradation whereas the role of cathepsin L is restricted to lysosomes.

A dihydropyridine-sensitive calcium channel in rodent osteoblastic cells

Rat osteogenic sarcoma cells (UMR 106-01) and normal rat trabecular bone osteoblasts (ROB) were studied using the whole cell version of the patch clamp technique to determine the existence of calcium (Ca2+) channels. Pipette and bath solutions were designed to separate Ca2+ channel currents from other voltage-dependent currents, and Ba2+ was used as the charge carrier. In both UMR 106-01 and ROB cells, a Ba2+ current was measured, which expressed the characteristics of an L-channel, such as activation range, dihydropyridine sensitivity, and little or no inactivation. In some cases, this channel was detectable only with BAY-K-8644 in the bath solution. The dihydropyridine agonist increased the current intensity and shifted the peak inward current to more negative potentials. This study, confirming previous observations, demonstrates the existence of a Ca2+ channel in both transformed and normal osteoblastic cells.

Electron microscopy of osteocytes and the pericellular matrix in rat trabecular bone.

The fine structure of the osteocytes and of the immediately adjacent bone matrix has been studied in the jaws of young rats demineralized with EDTA. The events marking the life cycle of the cell and their effects on the pericellular bone substance have been grouped into 3 phases. 1. The formative period, where the osteocyte resembles an osteoblast but shows a gradual decrease in the amount of endoplasmic reticulum and in the size of the Golgi complex. 2. The beginning of resorption (osteocytic osteolysis) which is characterized by a further decrease of the secretory organelles and the jagged appearance of the perilacunar border. Later in this phase there is further development and activity of the lysosomes resulting in increased widening of the lacuna and accumulation in the lacuna of fibrillar and flocculent material. 3. The eventual degeneration and death of the cell. No evidence of regeneration (“osteoplasia”) has been observed.