The interaction between receptor activator of nuclear factor κB ligand (RANKL) and osteoprotegerin (OPG) plays a dominant role in osteoclastogenesis. As both proteins are produced by osteoblast lineage cells, they are considered to represent a key link between bone formation and resorption. In this study, we investigated the expression of RANKL and OPG during bone remodeling in vivo to determine the relationship between osteoclastogenic stimulation and osteoblastic differentiation. Total RNA was prepared from rat femurs after marrow ablation on days 0, 3, 6, and 9. The temporal activation patterns of osteoblast-related genes (procollagen α1 (I), alkaline phosphatase, osteopontin, and osteocalcin) were examined by Northern blot analysis. An appreciable increase in the expression of these osteoblast markers was observed on day 3. The peak increase in gene expression was observed on day 6 followed by a slight reduction by day 9. Real-time PCR analysis showed that the OPG mRNA expression was markedly upregulated on day 6 and slightly decreased on day 9. In contrast, RANKL mRNA expression was increased by more than 20-fold on day 9. The RANKL/OPG ratio, an index of osteoclastogenic stimulation, peaked on day 9. Histological analysis showed that RANKL and OPG immunoreactivity were predominantly associated with bone marrow cells. The expression of bone formation markers was activated in the bone formation phase, followed by the stimulation of RANKL/OPG expression in the bone resorption phase, which confirmed that these molecules are key factors linking bone formation to resorption during bone remodeling.

The effects of basic fibroblast growth factor (bFGF) on osteogenic differentiation in-vivo were investigated using a rat bone marrow ablation model. bFGF was infused directly into rat femora for 6 days after bone marrow ablation. The contralateral femur was infused with vehicle only and used as control. Bone formation was induced in the rat femoral cavity, and the gene expression of osteoblast markers was examined. Treatment with bFGF at 50 and 100ng/day significantly enhanced the mRNA levels of osteopontin compared with the levels in the control leg, with increases of 25% and 24%, respectively. In contrast, bFGF infusion at 50 ng/day provoked a significant (nearly 20%) inhibition of expression for type I collagen. Infusion of bFGF at a higher dose exhibited an inhibitory tendency for bFGF action on gene expression. There were no significant changes in alkaline phosphatase and osteocalcin mRNA levels in response to any dose of bFGF. The findings presented here suggest that bFGF modulates osteogenic differentiation in-vivo and may play an important role in the process of bone remodeling.

In order to establish the cellular basis for using growth factors as possible therapeutic agents for the age-dependent deficit in bone formation activity, we examined the individual and combined effects of IGF-I and/or platelet-derived growth factor (PDGF) on the gene expression of osteoblast-related markers in male rats.The expression of osteoblast markers was examined in the femurs of adult and old rats following marrow ablation, which amplifies gene expression activity. The mRNA levels of collagen(alpha1) (I) (COLI), alkaline phosphatase (AP), osteopontin (OP) and osteocalcin (OC) were significantly lower in the old as compared with the adult rats. To determine whether growth factors can abolish the age-related deficits in mRNA expression in old bone, PDGF and/or IGF-I were infused directly into the right femur for 5 days following marrow ablation. The contralateral femur was infused with vehicle only and used as a control. PDGF stimulated the expression of OP mRNA in both adult and old rats, whereas COLI, AP and OC mRNAs were not affected. IGF-I infusion did not have a significant effect on mRNA expression in adult rats. In contrast, treatment with IGF-I significantly enhanced the mRNA levels of COLI, AP and OP in old rats. To examine whether the combination of both factors could affect the expression of osteoblast markers synergistically, PDGF and IGF-I were infused together. In adult bones, the combined treatment with PDGF and IGF-I caused a slight increase in the level of OP gene expression but no change in AP, OC or COLI genes. Although neither IGF-I nor PDGF alone was effective in stimulating the expression of OC, the combined treatment in old bones enhanced OC expression significantly. The expression of COLI, AP and OP was also stimulated, but the stimulation was no different from that of IGF-I alone. In PDGF plus IGF-I treatment with a high dose, no dose-response effects were observed.Within the limits of the present study, it is suggested that IGF-I and, to a much lesser extent, PDGF may partially restore the deficit in the expression of osteoblast markers in old bones, and that the combination of both factors is slightly better than IGF-I alone in stimulating OC expression.

Local and systemic expression of insulin-like growth factor-I (IGF-I) during bone formation was studied using the rat bone marrow ablation model. The temporal expression pattern of IGF-I mRNA in rat femurs was examined. The IGF-I mRNA level was enhanced rapidly after ablation reaching a level threefold greater than basal by day 3 (P < 0.01) and declined to basal or below basal level by day 5. Histological analysis showed that IGF- I immunoreactivity was predominantly associated with the mesenchymal cells at the bone/connective tissue interface and osteoblastic cells at active sites of bone formation. Serum level of IGF-I increased 50 and 130%, respectively (P < 0.005), over the basal level at days 3 and 6. We also investigated the systemic expression of IGF-I in liver and kidney. In contrast, hepatic IGF-I gene expression decreased 37 and 48%, respectively, at days 3 and 6 after marrow ablation (P < 0.001). Kidney IGF-I mRNA levels also fell 13 and 27%, respectively, at days 3 and 6 (P < 0.005). The present findings suggest that locally produced IGF-I during bone formation may not only serve as an autocrine/paracrine factor but also influence systemic expression of IGF-I in other organs.

Previously we found that PGE(2) 3 mg/kg in 20-month-old male rats induced massive bone formation mainly by modeling dependent bone gain in cortical bone. It is not known whether cancellous bone will respond similarly; thus, we evaluated the effect of PGE(2) on cancellous bone of the same aged rats. Thirty-four 20-month-old Wistar male rats were given PGE(2) (3 mg/kg/day) or vehicle subcutanously for 10 and 30 days. Double fluorescent labels were injected 9 and 2 days prior to the sacrifice. Histomorphometry was performed on 1% toluidine blue stained and unstained sagittal sections of lumbar vertebral bodies. The results demonstrated that 10-day PGE(2) treatment increased osteoprogenitor cells, osteoblasts (x2-fold), osteoid (x4.5-fold), woven bone formation (0.04%), and 40% more trabecular area; it stimulated modeling (x2-fold) and remodeling-dependent (x1.5-fold) bone formation with increase of mineralization lag time (MLT, x 7.5-fold). Thirty-day treatment sustained increases in osteoblast numbers, modeling, and remodeling-dependent bone formation and further stimulated woven bone formation (6.6%), turnover (x3-fold), and trabecular area and number (x2-fold). Osteoprogenitor cells were undetectable along with 70% less osteoid area compared with 10-day treatment but still was 1.5-fold higher than aging controls. MLT returned to aging control level. It was concluded that the aged cancellous bone of 20-month-old male Wistar rat retains a capability of responding to the anabolic effect of PGE(2). Osteoblastogenesis and enhanced modeling and remodeling-dependent woven or lamellar formation contribute to this anabolic action. Bone formation differed in that the endocortical surface of cortical bone was stimulated mainly by modeling whereas both modeling and remodeling-dependent bone gain were equally stimulated at the trabecular surface of the lumbar vertebral body.

The present study examined the early effects of prostaglandin (PG)E 2 on proximal tibial metaphyses of 20-month-old Wistar male rats. PGE 2 was given to intact rats for 10 and 30 days at 3 mg/kg/day. After multiple in vivo fluorochrome labeling, undecalcified longitudinal sections were subjected to analysis of bone histomorphometry and classification of the contour of the cement line in bone formation units. The latter was used to classify bone formation units into modeling, remodeling and uncertain units. After 10 days of treatment, there was a 2% increase in woven bone formation with the appearance of osteoprogenitor cells and increases in the number of osteoblasts (649%) and osteoid (375%) surfaces. Remodeling and modeling units increased by 56% and 429%, respectively. After 30 days of treatment, there was an increase of 212% of total trabecular bone mass, 60% of which was woven bone. In addition, there were increases in labeling surface (147%), mineral apposition rate (760%), bone formation rates tissue area (BFR/T.Ar, 1920%; BFR/B.Pm, 343%), and bone turnover (BFR/B.Ar, 426%). Osteoblasts and osteoid production at 30 days were 29% and 58% less than at 10 days post-treatment. Modeling and remodeling activity did not differ from that seen at 10 days. In addition, PGE 2 treatment tended to stimulate the closing of growth plates and decrease the fatty marrow area. We conclude that the aged skeleton was able to respond vigorously to PGE 2 treatment. Massive osteoprogenitors cells, and osteoid and osteoblast formations were observed within 10 days, and dramatic woven and lamellar bone formation was seen at 30 days post-treatment. The anabolic effects were driven mainly by modeling.

ABSTRACT: In the current study, we examined the effects of minocycline on the osteopenia of ovariectomized (OVX) aged rats using the marrow ablation model. This injury induces rapid bone formation followed by bone resorption in the marrow cavity. Old female rats were randomly divided into five groups: sham, OVX, OVX+minocycline (5‐15 mg/day, orally), OVX+17β‐estradiol (25 μg/day, subcutaneously), and OVX+both agents. Rats were OVX, treated with minocycline and/or estrogen, followed by marrow ablation. Bone samples were collected 16 days post‐marrow ablation. X‐ray radiography of bones operated on showed that treatment of OVX old rats with minocycline increased bone mass in diaphyseal region. Diaphyseal bone mineral density (BMD) was measured by DEXA scan. Diaphyseal BMD of OVX rats was increased 17‐25% by treatment with 5‐15 mg of minocycline or 17β‐estradiol. The effects of minocycline and estrogen treatments on the expression of osteoblast and osteoclast markers were also examined. Northern and dot blot analysis of RNA samples showed that treatment of OVX aged rats with minocycline increased the expression of type I collagen (COL I) (49%) and decreased that of interleukin‐6 (IL‐6) (31%). In contrast, estrogen treatment decreased the expression of interleukin‐6 (IL‐6) (39%), carbonic anhydrase II (CA II) (36%), and osteopontin (OP) (37%). Neither minocycline nor 17β‐estradiol had an effect on the expression of osteocalcin (OC) and alkaline phosphatase (AP). To elucidate the mechanism by which minocycline prevented the loss of bone in OVX aged rats, we examined the colony‐formation potential of bone marrow stromal cells in ex vivo cultures. Minocycline stimulated the colony‐forming efficiency of marrow stromal cells derived from old animals. We have therefore concluded that the modest increase in BMD noted in OVX aged rats, in response to minocycline treatment, may be due to a change in bone remodeling that favors bone formation; and the anabolic effect of minocycline is likely due to its effect on the expression of COL I and/or the metabolism of osteoprogenitor cells.

Rat osteoprogenitor cells were used to examine the effects of bFGF on DNA synthesis and the expression of osteoblast (OB)-related genes. bFGF, as low as 0.1 ng/ml, stimulated DNA synthesis. bFGF also increased the mRNA level of osteopontin (OP) and decreased that of type I collagen (COL I). When cultures were grown in dexamethasone (DEX) to induce OB lineage commitment, the expression of COL I, alkaline phosphatase (AP) and OP was greatly enhanced. Subsequent incubation with bFGF partially negated the stimulatory effect of DEX on AP and COL I mRNAs. bFGF also inhibited the expression of osteocalcin mRNA in cells grown in 1,25(OH)2D3 and DEX. Combined effects of bFGF with IGF-I or PDGF on DNA synthesis and OP expression were examined. bFGF+IGF-I, but not bFGF+PDGF, was more effective than PDGF alone. By comparing cells from adult and old animals, we found that bFGF-induced mitogenic activity was reduced significantly with age. In contrast, the effect of bFGF on the expression of OB genes was not significantly altered by age. These findings suggest that bFGF plays a dual role as a local positive and negative regulator on proliferation and osteogenic lineage expression, respectively, in osteoprogenitor cells, and that the mitogenic activity in response to bFGF was impaired in aging.

The effect of oral minocycline on osteopenia in ovariectomized (OVX) old rats was examined in this study. Rats were divided into 4 groups: sham-operated, OVX followed by treatment with vehicle, minocycline, or 17β-estradiol. The treatment was initiated one day after OVX and proceeded for 8 wks. OVX reduced bone mineral density (BMD) in the whole femur and in the femoral regions that are enriched in trabecular bone. Treatment with minocycline or estrogen prevented a decrease in BMD. Femoral trabecular bone area, trabecular number, and trabecular thickness were reduced, and trabecular separation was increased by OVX. Treatment with minocycline or estrogen abolished the detrimental effects induced by OVX. OVX also reduced indices that reflect the interconnectivity of trabecular bone, and the loss of trabecular connectivity was prevented by treatment with minocycline or estrogen. Based on the levels of urinary pyridinoline, we showed that the effect of estrogen, but not minocycline, was primarily through its inhibitory effect on bone resorption. Analysis of bone turnover activity suggests that OVX increased parameters associated with bone resorption (eroded surface) and formation (osteoid surface, mineralizing surface, mineral apposition rate, and bone formation rate). Treatment with minocycline reduced bone resorption modestly and stimulated bone formation substantially. In contrast, treatment with estrogen drastically reduced parameters associated with both bone resorption and formation. We have concluded that oral minocycline can effectively prevent the decrease in BMD and trabecular bone through its dual effects on bone resorption and formation.

Previously, we showed that the age-dependent deficit in bone formation activity can be attributed in part to a decline in local expression of insulin-like growth factor I (IGF-I) and altered mitogenic response of old osteoprogenitor cells to IGF-I. To establish the cellular basis for using IGF-I as a possible therapeutic agent for osteoporosis, we examined the effect of locally infused (50 ng/day for 14 days) on the expression of osteoblast-related genes in femurs of old rats. Northern and dot blot analyses showed that the expression of procollagen (I), osteopontin, alkaline phosphatase, and osteocalcin was increased 0.4- to 1.5-fold in IGF-I-treated femurs as compared with control femurs. Histomorphometric analyses were carried out in parallel experiments to assess the changes in bone remodeling activity. Trabecular bone volume, trabecular number, and trabecular thickness were increased 56%, 29%, and 23%, respectively, whereas trabecular separation was reduced 26% by IGF-I treatment. IGF-I treatment increased significantly the osteoid volume, osteoid surface, osteoblast number, and osteoblast surface. Mineralizing surface and mineral apposition rate, kinetic indices of bone formation, were also stimulated by IGF-I treatment. The bone formation rate was stimulated 81% in IGF-I-treated femurs as compared with control femurs. In contrast, eroded surface and osteoclast surface, parameters associated with bone resorption, were not affected by IGF-I treatment. These findings suggest that local administration of IGF-I into femurs of old rats can stimulate the expression of matrix proteins and improve trabecular bone status by stimulating bone formation without any appreciable effect on bone resorption.

In the current study, we examined the effects of minocycline, on the osteopenia of ovariectomized aged rats. Old female rats were randomly divided into five groups: sham, ovariectomized control and ovariectomized treated with minocycline, 17beta-estradiol, or both agents. Bone samples were collected 8 wk after the treatment. Ovariectomy reduced bone mineral density of the whole femur and at the condylar, distal metaphyseal and head-neck-trochanter regions 10%-19% and the loss of bone density was prevented by treatment with minocycline or 17beta-estradiol. Histomorphometric analysis of distal femur showed ovariectomy reduced the trabecular bone area, the trabecular bone number, trabecular bone thickness and increased the trabecular bone separation. The microanatomic structure of trabecular bone also showed that the number of nodes, node to node, cortical to node, node to free end was reduced by ovariectomy. Treatment with minocycline attenuated the effect of ovariectomy on trabecular bone in aged animals. In contrast, cortical bone was not affected by ovariectomy or minocycline treatment. The effect of minocycline on bone turnover was also examined. Minocycline increased osteoid surface, mineralizing surface, mineral apposition rate, bone formation rate and reduced eroded surface. We have therefore concluded that the modest increase in bone mineral density and the improvement in the trabecular bone status noted in minocycline treated ovariectomized aged rats is likely due to an increase in bone formation coupled with a decrease in bone resorption.

The age-related deficit in the dose response of osteoprogenitor cells to IGF-I was further investigated. As expected, the effective dose, but not the maximal effect, was shifted two orders of magnitude higher in old cells. In this paper, we examined whether this age-deficit can be attributed to an alteration in the expression and binding kinetics of IGF-I receptor. We showed that the levels of IGF-I receptor mRNA in cells, estimated by RT-PCR, were not significantly altered with age. Scatchard analysis showed that there were no significant differences in Kd and Bmax in cells from the two age groups. In a parallel study, we also showed that the expression of osteoblast phenotype markers was stimulated by IGF-I. However, no apparent differences in dose response curve were observed between two age groups. These results suggest that defect(s) in cell proliferation in aging may occur specifically in the signal transduction pathway between the receptor and the mitogenic response but not in the pathway associated with phenotype expression.

The expression of insulin-like growth factor-I (IGF-I), interleukin-6 (IL-6), and transforming growth factor-beta 1 (TGF-beta 1) mRNA in rat femurs was examined following marrow ablation. Northern blot analysis showed multiple transcripts of IGF-I, a major transcript of 1.3 kb and a minor one of 2.4 kb for IL-6 and a single band of 2.5 kb for TGF-beta 1, respectively. Examination of the temporal activation pattern showed IGF-I expression peaked at day 3 (150% over the basal level) after injury and preceded the maximal expression of procollagen alpha 1(I), osteopontin, alkaline phosphatase, and osteocalcin mRNAs. This suggests that IGF-I is involved mainly in osteoblast development and bone formation. In contrast, IL-6 expression was elevated between days 3 and 9 (45-60% over the basal level). The sustained elevation of IL-6 expression at day 9 is consistent with the role for this cytokine in the development of osteoclasts and bone resorption. The expression of TGF-beta 1 was not altered up to day 9 after marrow ablation. While the temporal expression patterns of IGF-I and IL-6 mRNA did not differ between adult and old rats, the maximal level of IGF-I mRNA at day 3 was 72% higher in adult as compared to old bones. In contrast, the peak level of IL-6 mRNA at days 6-9 was 45% higher in old as compared to adult bones. Although the level of TGF-beta 1 mRNA did not change following marrow ablation, levels of TGF-beta 1 were consistently higher in old rats. Our results suggest that the impaired bone formation and elevated bone resorption in aged animals may be due in part to the reduced expression of IGF-I and an overexpression of IL-6 in old bone.

The effects of platelet-derived growth factor (PDGF) on DNA synthesis and mRNA expression of osteoblast markers in marrow stromal cells derived from adult (6 months) and old (24 months) rats were examined. Treatment of stromal cells from adult rats with dexamethasone induced the appearance of osteoblast-like cells. PDGF partially also inhibited the differentiation of stromal cells induced by dexamethasone. In cultures of serum-starved stromal cells, PDGF stimulated [3H]-thymidine incorporation into DNA in a dose-dependent manner with a maximum stimulation of 15-fold at 500 ng/ml. By comparison, insulin-like growth factor (IGF-I) has a small effect on [3H]-thymidine incorporation. The effect of PDGF and IGF-I on DNA synthesis was additive. Treatment of the confluent stromal cells from adult rats with PDGF increased the mRNA level of osteopontin fourfold without any significant effect on alkaline phosphatase and type I collagen mRNAs. In contrast, dexamethasone stimulated the mRNA expression of alkaline phosphatase, type I collagen, and osteopontin 2.1-, 2.3-, and 14-fold, respectively. Addition of PDGF to dexamethasone-treated cells failed to induce any further increase in osteopontin expression whereas the expression of alkaline phosphatase and type I collagen was partially reduced. The expression of osteocalcin mRNA was negligible in stromal cells but stimulated several fold by dexamethasone and 1,25(OH)2D3. PDGF inhibited drastically the elevation of osteocalcin mRNA. In contrast, IGF-I stimulated type I collagen expression 100% without any appreciable effect on the expression of osteopontin and alkaline phosphatase. The stimulatory effect of PDGF on osteopontin expression was augmented by IGF-I. Furthermore, PDGF attenuated the stimulatory effect of IGF-I on type I collagen expression. The responses of cultured cells from old rats to growth factors were also examined. PDGF or PDGF plus IGF-I increased [3H]-thymidine incorporation in stromal cells from old rats but to a lesser extent. However, PDGF was equally effective in stimulating osteopontin expression in cells from both adult and old rats. We concluded that PDGF is a potent mitogen but that the response of stromal cells from old rats is impaired. In addition, PDGF stimulates osteopontin expression in stromal cells and this effect is not age dependent.

Aging bone shows a progressive decline in mass and strength. Previous studies have suggested that bone marrow stem cells are reduced with aging and that this could be responsible, in part, for age-associated bone deficits. We measured the number of osteoprogenitor cells present in the bone marrow from adult and aged rats as well as their ability to differentiate in vitro and to form bone in vivo. We found that the number of adherent colony-forming cells was significantly lower (65%) in marrow cells isolated from aged compared with adult rats. Furthermore, 88% of the colonies obtained from aged rats were alkaline phosphatase (AP) positive, whereas virtually all the colonies from adult rats were positive. The addition of dexamethasone to the culture medium decreased the proliferation of the adherent cells and reduced the number of colonies obtained from both adult and aged bone marrow, all of which were AP positive. No significant differences were found in the expression of certain major bone cell marker genes as a function of donor age. However, dexamethasone treatment increased expression of osteopontin (OP) by fivefold. Adult stromal cells not treated with dexamethasone and implanted subcutaneously in recipient rats exhibited about 10-fold greater formation of bone compared with cells from aged rats. In contrast, dexamethasone-treated cells exhibited high levels of bone formation, irregardless of donor age or the age of the recipient into which the cells were grafted. These studies are consistent with a deficit of osteoprogenitor cells in the bone marrow site as a contributing, perhaps correctable factor in the decline in bone repair and bone mass with age.

Previously, we reported that uptake of calcium into isolated duodenal cells and duodenal brush border membrane vesicles decreased in senescence. Decreases in duodenal 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] receptor number and 9k vitamin D-dependent calcium binding protein (CaBP) were also observed in aged rats. In this study, we examined the steady state mRNA levels of duodenal 1,25-(OH)2D3 receptor and CaBP in both adult (6-month-old) and old (24-month-old) rats. We identified one major band of 4.4 kb for 1,25-(OH)2D3 receptor mRNA. The size of the transcript was not affected by age. The content of 1,25-(OH)2D3 receptor mRNA (normalized with poly(A)+RNA) decreased 23% in the aged rat as compared to the adult rat. The expression of CaBP was also examined. A single band of 0.6 kb was observed for CaBP mRNA. The size of CaBP mRNA was not altered with age. However, the abundance of CaBP mRNA (normalized with poly(A)+RNA) was reduced 20% in the senescent rat. Thus, the results in the present study were consistent with our previous findings that the number of 1,25-(OH)2D3 receptors and the level of CaBP declined in the aged rat. However, the precise mechanism leading to the age-related deficit in mRNA expression remains to be elucidated.

The in vivo response of bone to IGF-I infusion in a marrow ablation model and the effect of IGF-I on bone marrow stromal cells in vitro was evaluated. IGF-I (25 ng/day), infused directly into femur, stimulated the expression of alkaline phosphatase, procollagen ∝1 (I) and osteopontin mRNA, while osteocalcin mRNA was not affected. The dose dependency to IGF-I was bi-phasic, with stimulation at 25 and 50 ng but not at 150 ng/day. The effect of IGF-I was observed in the aged but not in the adult rat femur. However, the elevated mRNA levels in old bones with IGF-I treatment were still below those observed in adult bones. The effect of IGF-I was also examined in cultured stromal cells. IGF-I (50 ng/ml) stimulates the expression of alkaline phosphatase, procollagen ∝1 (I), osteopontin and osteocalcin mRNA in stromal cells from both adult and old rats. These results suggest that the lack of response of adult bone to IGF-I in vivo was not due to the impaired response of the stromal cells to IGF-I. Differences in the responses of stromal cells from adult and old animals were noted. In the presence of serum (10%), stromal cells from adult rats were stimulated to synthesize DNA at lower levels of IGF-I than stromal cells from old animals. Our results show that IGF-I can stimulate mRNA expression of osteoblast markers in vivo in aged rats in a marrow ablation model and enhance DNA synthesis and gene expression in cultured marrow stromal cells from old rats. Thus, it is possible that exogenous IGF-I could be beneficial in treating age-associated osteopenia.