Osteoblast Collagen Synthesis Research Articles

Preosteoclast plays a pathogenic role in syndesmophyte formation of ankylosing spondylitis through the secreted PDGFB — GRB2/ERK/RUNX2 pathway

ObjectivesAnkylosing spondylitis (AS) is a chronic inflammatory disease that mainly affects the sacroiliac joint and spine. However, the real mechanisms of immune cells acting on syndesmophyte formation in AS are not well identified. We aimed to find the key AS-associated cytokine and assess its pathogenic role in AS.MethodsA protein array with 1000 cytokines was performed in five AS patients with the first diagnosis and five age- and gender-matched healthy controls to discover the differentially expressed cytokines. The candidate differentially expressed cytokines were further quantified by multiplex protein quantitation (3 AS-associated cytokines and 3 PDGF-pathway cytokines) and ELISA (PDGFB) in independent samples (a total of 140 AS patients vs 140 healthy controls). The effects of PDGFB, the candidate cytokine, were examined by using adipose-derived stem cells (ADSCs) and human fetal osteoblast cell line (hFOB1.19) as in vitro mesenchymal cell and preosteoblast models, respectively. Furthermore, whole-transcriptome sequencing and enrichment of phosphorylated peptides were performed by using cell models to explore the underlying mechanisms of PDGFB. The xCELLigence system was applied to examine the proliferation, chemotaxis, and migration abilities of PDGFB-stimulated or PDGFB-unstimulated cells.ResultsThe PDGF pathway was observed to have abnormal expression in the protein array, and PDGFB expression was further found to be up-regulated in 140 Chinese AS patients. Importantly, PDGFB expression was significantly correlated with BASFI (Pearson coefficient/p value = 0.62/6.70E − 8) and with the variance of the mSASSS score (mSASSS 2 years − baseline, Pearson coefficient/p value = 0.76/8.75E − 10). In AS patients, preosteoclasts secreted more PDGFB than the healthy controls (p value = 1.16E − 2), which could promote ADSCs osteogenesis and enhance collagen synthesis (COLI and COLIII) of osteoblasts (hFOB 1.19). In addition, PDGFB promoted the proliferation, chemotaxis, and migration of ADSCs. Mechanismly, in ADSCs, PDGFB stimulated ERK phosphorylation by upregulating GRB2 expression and then increased the expression of RUNX2 to promote osteoblastogenesis of ADSCs.ConclusionPDGFB stimulates the GRB2/ERK/RUNX2 pathway in ADSCs, promotes osteoblastogenesis of ADSCs, and enhances the extracellular matrix of osteoblasts, which may contribute to pathological bone formation in AS.

Chronic exposure to environmentally relevant concentration of fluoride impairs osteoblast's collagen synthesis and matrix mineralization: Involvement of epigenetic regulation in skeletal fluorosis

Globally, 200 million people are suffering from toxic manifestations of Fluoride(F), dental and skeletal fluorosis; unfortunately, there is no treatment. To unravel the pathogenesis of skeletal fluorosis, we established fluorosis mice by treating environmentally relevant concentration of F (15 ppm NaF) through drinking water for 4 months. As in skeletal fluorosis, locomotor disability, crippling deformities occur and thus, our hypothesis was F might adversely affects collagen which gives the bone tensile strength. This work inevitably had to be carried out on osteoblast cells, responsible for synthesis, deposition, and mineralization of bone matrix. Isolated osteoblast cells were confirmed by ALP activity and mineralized nodules formation. Expression of collagen Col1a1, Col1a2, COL1A1 was significantly reduced in treated mice. Further, a study revealed the involvement of epigenetic regulation by promoter hypermethylation of Col1a1; expressional alterations of transcription factors, calcium channels and other genes e.g., Cbfa-1, Tgf-β1, Bmp1, Sp1, Sp7, Nf-Kb p65, Bmp-2, Bglap, Gprc6a and Cav1.2 are associated with impairment of collagen synthesis, deposition and decreased mineralization thus, enfeebling bone health. This study indicates the possible association of epigenetic regulation in skeletal fluorosis. However, no association was found between polymorphisms in the Col1a1 (RsaI, HindIII) and Col1a2 (RsaI, HindIII) genes with fluorosis in mice.

Defect-related luminescent microstructured hydroxyapatite promote bone regeneration through nucleating effect

Non-nano scaled hydroxyapatite (HAP) particles cannot enter the cells, but they are also wildly used in tissue engineering for their excellent bone regeneration. We synthesized a defect-related luminescent micro-scale hydroxyapatite particles (S3) and investigated the effect of S3 during bone regeneration. S3 promoted the formation of mineralized nodules and collagen synthesis of osteoblasts (OBs). Micro-scaled S3 couldn't enter into OBs and couldn't change the Ca2+ concentration of the medium. During the cell differentiation, the location of S3 was tracked by its defect-related luminescence in vitro. Extracellular S3 particles become the nucleation events which promote bone regeneration. The results suggest that micro-scale HAP promoted bone regeneration through extracellular pathways. This result also can explain the reason why hydroxyapatite covered tissue scaffold is more suitable for bone reconstructing.

Quantitative in situ time-series evaluation of osteoblastic collagen synthesis under cyclic strain using second-harmonic-generation microscopy.

.The aim of this study is to evaluate the osteoblastic collagen synthesis under mechanical stimulation using second-harmonic-generation (SHG) microscopy. We apply SHG microscopy to monitor the collagen fibers synthesized by osteoblast-like cells (MC3T3-E1) without the need for fixation and staining. To quantitatively evaluate the influence of mechanical stimulation on osteoblastic collagen synthesis, we compare SHG images of osteoblast-synthesized collagen fibers with and without a cyclic stretch stimulus applied using a lab-made stretching device. We acquire SHG images every 7 days for 3 weeks at different stimulus conditions (5 min/day and 3 h/day with a strain magnitude of 5% and a frequency of 0.5 Hz). Image analysis of the average SHG intensity indicates that the amount of osteoblastic collagen synthesis is significantly enhanced by the cyclic stretch compared with the nonstretched condition, while there is no significant difference between the two mechanical stimulation conditions. Furthermore, the maturity of the collagen fibers in the early stage of bone formation is not affected by the mechanical stimulation. The results can be used in bone regenerative medicine to apply feedback control of collagen synthesis by artificial stimulation.

Activation of JNK signaling in osteoblasts is inversely correlated with collagen synthesis in age-related osteoporosis

The age-related reduction in the function of osteoblasts plays a central role in the pathogenesis of bone loss and osteoporosis. Collagen synthesis is a primary function of differentiated osteoblasts, however, the mechanisms for age-related changes in collagen synthesis in human osteoblasts remain elusive.We use Gene Ontology (GO) analysis and Gene Set Enrichment Analysis (GSEA) analysis to exploit the transcriptional profiles of osteoblasts from young and old donors. A panel of collagen members was downregulated in aged osteoblasts, including COL12A1, COL5A1, COL5A3, COL8A1 and COL8A2. Co-expression analysis followed by GO analysis revealed that oxidoreductase activity and kinase activity were inversely correlated with collagen synthesis in osteoblasts. GESA analysis further showed that JNK signaling was upregulated in aged osteoblasts. Consistently, MAP3K4 and MAP4K2, upstream of JNK, were also increased in aged osteoblasts. Moreover, expression levels of MAP3K4 were significantly inversely correlated with levels of the collagen genes. Those transcriptomic results were further verified by examining clinical specimens of osteoporosis by immunohistochemistry.These results provide transcriptomic evidence that deregulated JNK signaling may impair collagen synthesis in osteoblasts and imply a therapeutic value of JNK inhibitors for treating osteoporosis and preventing skeletal aging by counteracting the age-related reduction in the function of osteoblasts.

Concentration of several osteotropic growth factors, markers of osteogenesis and biologically active molecules in the blood serum of patients with congenital pseudarthrosis of tibia during orthopaedic treatment with combined technologies

Congenital pseudarthrosis of tibia is a genetic, systemic pathology with impaired bone remodeling and unknown pathogenetic mechanisms. Orthopaedic treatment of the disease can fail in some cases. The process of bone remodeling is known to occur under control of local and systemic growth factors, and we sought to explore several osteotropic growth factors, markers of osteogenesis and biologically active molecules in the blood serum of patients with congenital pseudarthrosis of tibia. The study included 12 patients with congenital pseudarthrosis of tibia and anatomical shortening of 2.5±1.1 cm. The' age of patients ranged from 7 years to 18 years. Blood serum was used for enzyme immunoassay analysis. The own blood serum levels of 103 conditionally healthy individuals (of mean age of 13.0±0.27 years) were considered as the norm. Greater changes in the concentration were detected among vascular endothelial and transforming growth factors. The patients showed imbalance in serum TGF, low reparative potential of bone tissue due to osteoclast activation prevailing over differentiation of osteoblasts, progenitor and mesenchymal cells. Dynamics in serum concentration of IGF at the time of frame removal indicated to terminating osteoblast activation and collagen synthesis and concomitant active bone restructuring.

微小振動とストレッチを組み合わせた刺激による骨芽細胞のコラーゲン産生促進

微小振動とストレッチを組み合わせた刺激による骨芽細胞のコラーゲン産生促進

Antiosteoporotic and antioxidant activities of diterpenoids from the Vietnamese soft corals Sinularia maxima and Lobophytum crassum

Marine organisms possess bioactive potential with tremendous pharmaceutical promise. The purpose of the present study was to investigate the antioxidant (peroxyl radical-scavenging and reducing capacities) and antiosteoporotic activities of isolated constituents (1–26) from the soft corals Sinularia maxima and Lobophytum crassum on pre-osteoblastic MC3T3-E1 cells and pre-osteoclastic RAW 264.7 cells. Among the isolated compounds, 7, 9, 17, 18, and 20 (10.0 μM) exhibited significant peroxyl radical-scavenging capacity, with TE values ranging from 4.00 ± 0.14 to 9.06 ± 0.33 μM. In addition, compounds 1, 5, 7, 20, and 24 significantly stimulated the differentiation of pre-osteoblastic MC3T3-E1 cells by increasing collagen synthesis and/or mineralization functions of osteoblasts, while compounds 1, 5–8, 12, 14–22, 24, and 26 showed significant inhibition of TRAP in NF-κB ligand-induced osteoclastic RAW 264.7 cells, with values ranging from 99.11 ± 1.36 to 61.54 ± 1.61 %. These results indicate that the soft corals S. maxima and L. crassum are excellent sources among the antioxidant and antiosteoporotic marine invertebrates.

Glucose Uptake and Runx2 Synergize to Orchestrate Osteoblast Differentiation and Bone Formation

The synthesis of type I collagen, the main component of bone matrix, precedes the expression of Runx2, the earliest determinant of osteoblast differentiation. We hypothesized that the energetic needs of osteoblasts might explain this apparent paradox. We show here that glucose, the main nutrient of osteoblasts, is transported in these cells through Glut1, whose expression precedes that of Runx2. Glucose uptake favors osteoblast differentiation by suppressing the AMPK-dependent proteasomal degradation of Runx2 and promotes bone formation by inhibiting another function of AMPK. While RUNX2 cannot induce osteoblast differentiation when glucose uptake is compromised, raising blood glucose levels restores collagen synthesis in Runx2-null osteoblasts and initiates bone formation in Runx2-deficient embryos. Moreover, RUNX2 favors Glut1 expression, and this feedforward regulation between RUNX2 and Glut1 determines the onset of osteoblast differentiation during development and the extent of bone formation throughout life. These results reveal an unexpected intricacy between bone and glucose metabolism.

Sumoylation of the Tumor Suppressor Promyelocytic Leukemia Protein Regulates Arsenic Trioxide-Induced Collagen Synthesis in Osteoblasts

Background/Aims: Promyelocytic leukemia (PML) protein is a tumor suppressor that fuses with retinoic acid receptor-α (PML-RARα) to contribute to the initiation of acute promyelocytic leukemia (APL). Arsenic trioxide (ATO) upregulates expression of TGF-β1, promoting collagen synthesis in osteoblasts, and ATO binds directly to PML to induce oligomerization, sumoylation, and ubiquitination. However, how ATO upregulates TGF-β1 expression is uncertain. Thus, we suggested that PML sumoylation is responsible for regulation of TGF-β1 protein expression. Methods: Kunming mice were treated with ATO, and osteoblasts were counted under scanning electron microscopy. Masson's staining was used to quantify collagen content. hFOB1.19 cells were transfected with siRNA against UBC9 or RNF4, and then treated with ATO or FBS. TGF-β1, PML expression, and sumoylation were quantified with Western blot, and collagen quantified via immunocytochemistry. Results: ATO enhanced osteoblast accumulation, collagen synthesis, and PML-NB formation in vivo. Knocking down UBC9 in hFOB1.19 cells inhibited ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conversely, knocking down RNF4 enhanced ATO- and FBS-induced PML sumoylation, TGF-β1 expression, and collagen synthesis. Conclusion: These data suggest that PML sumoylation is required for ATO-induced collagen synthesis in osteoblasts.

Evaluation of the antioxidant and anti-osteoporosis activities of chemical constituents of the fruits of Prunus mume

The present study investigated the antioxidant and anti-osteoporosis activities of phytochemicals in the fruits of Prunus mume. From the methanol extract, three new acylated sucroses, mumeoses P–R (1–3), were isolated together with 20 known compounds (4–23). Compounds 1–3 showed potent peroxyl radical-scavenging activities and 12–19 showed both potent peroxyl radical-scavenging and reducing activities. The anti-osteoporosis activity was evaluated using murine pre-osteoblastic MC3T3-E1 cells and pre-osteoclastic RAW 264.7 cells. Compounds 2 and 3 (cis–trans isomers), 5, 7, 8, and 10 significantly stimulated the differentiation of pre-osteoblastic MC3T3-E1 cells to increase collagen synthesis or mineralization functions of osteoblasts, while compounds 5, 6, 9, 10, 12, 14–16, 18, 20, and 22 significantly suppressed tartrate-resistant acid phosphatase activity in receptor activator of nuclear factor-κB ligand-induced osteoclastic RAW 264.7 cells. These results indicated that the fruits of P. mume are an excellent source of antioxidant and anti-osteoporosis phytochemicals.

Identification and biological evaluation of flavonoids from the fruits of Prunus mume

This Letter describes the identification of potent antioxidant and anti-osteoporosis agents from the fruits of Prunus mume. From the methanol extract, a novel flavan dimer, characterized as 2β,3β-epoxy-5,7,4′-trihydroxyflavan-(4α→8)-epicatechin (1), was isolated along with five known flavonoids (2–6). Their structures were determined based on extensive spectroscopic analysis, including IR, HRESIMS, 1D- and 2D-NMR, and CD spectra. The antioxidant activities of compounds 1–6 were evaluated in terms of their peroxyl radical-scavenging (Trolox equivalent) and reducing capacities. All isolates showed potent peroxyl radical-scavenging and reducing activities at concentrations of 1–10μM. Among them, compounds 1 and 2 were the most active at 1μM. Anti-osteoporosis activities were investigated using both murine osteoblastic MC3T3-E1 cells and osteoclastic RAW 264.7 cells. Compounds 2, 3, and 6 significantly stimulated the differentiation of osteoblastic MC3T3-E1 cells to increase collagen synthesis or mineralization functions of osteoblasts. Compounds 1, 3, 4, and 6 significantly suppressed tartrate-resistant acid phosphatase (TRAP) activity in receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclastic RAW 264.7 macrophage cells.

Cell viability, collagen synthesis and cytokine expression in human osteoblasts following incubation with generated wear particles using different bone cements

In total hip arthroplasty, wear particles generated at articulating surfaces and interfaces between bone, cement and implants have a negative impact on osteoblasts, leading to osteolysis and implant loosening. The aim of this experimental study was to determine the effects of particulate wear debris generated at the interface between straight stainless steel hip stems (Exeter(®)) and three different bone cements (Palacos(®) R, Simplex™ P and Cemex(®) Genta) on cell viability, collagen synthesis and cytokine expression in human osteoblasts. Primary osteoblasts were treated with various concentrations of wear particles. The synthesis of procollagen type I and different cytokines was analysed, and markers for apoptosis and necrosis were also detected. The cytokine synthesis rates in the osteoblasts were initially increased and varied, depending on incubation time and particle concentration. Specific differences in the synthesis rates of interleukin (IL)‑6, IL-8, vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) were observed with the different bone cements examined. The negative effect of the particles on the synthesis of procollagen type I and increased rates of cell apoptosis and necrosis were observed with all three cements analysed. Our present data suggest that wear particles from the interface between the total hip stem and bone cement have a significant effect on viability, cytokine expression and collagen synthesis in human osteoblasts, depending on the bone cement used.

Periprosthetic strain magnitude-dependent upregulation of type I collagen synthesis in human osteoblasts through an ERK1/2 pathway

Human osteoblasts sense mechanical stimulation and synthesise type I collagen in periprosthetic osseointegration following total hip arthroplasty. However, the regulation of type I collagen synthesis by periprosthetic strain is unclear because the cellular-level strain magnitude remains unknown to date. Fortunately, the tissue-level strain in implanted femurs is measurable. According to the mechanism of strain amplification, the tissue-level strain was amplified 20 times to stretch human osteoblasts in this study. Elongation of 0.8-3.2% enhanced the mRNA level of type I collagen, whereas the release of procollagen type I C propeptide only increased at 2.4% and 3.2% elongation. Type I collagen expression increased with the activation of ERK1/2 phosphorylation in a strain-magnitude-dependent manner, whereas JNK and P38 were unaffected. The responses were completely inhibited by blocking the ERK1/2 pathway with U0126. The results indicate that type I collagen synthesis in human osteoblasts depends on the level of periprosthetic strain and ERK1/2 activation.

Hormonal regulation of bone growth and remodelling.

Many systemic and local hormones influence bone growth and remodelling. These include calcium regulating hormones, systemic growth regulators and local growth factors. Parathyroid hormone (PHT) is a potent stimulator of osteoclastic bone resorption and a direct inhibitor of osteoblastic collagen synthesis. However, intermittent low-dose PTH administration can increase bone formation in vivo. PTH may act indirectly via local factors. It has been shown to increase prostaglandin E2 (PGE2) and transforming growth factor beta (TGF-beta) release from bone. Both PGE2 and TGF-beta have complex effects on bone metabolism and are likely to be physiological regulators of bone remodelling. Oestradiol has been shown to inhibit bone resorption in vivo but not in vitro. While there is evidence for oestrogen receptors in cultured bone cells, the effect could still be indirect. Oestradiol can inhibit bone PGE2 release in an in vivo-in vitro model in the rat. Glucocorticoids are potent inhibitors of bone formation and inhibit PGE2 and interleukin 1 production both in vivo and in vitro. While many regulatory factors affect prostaglandin production in bone, the complex effects of PGE2 on bone metabolism make it difficult to predict the ultimate response. The major effects of PGE2 are stimulation of bone formation and resorption and an increase in bone turnover. However, opposite effects can occur at certain times and concentrations. Interactions among these factors could explain some physiological, pathological, and therapeutic responses in skeletal tissue.

ATF4 mediation of NF1 functions in osteoblast reveals a nutritional basis for congenital skeletal dysplasiae

The transcription factor ATF4 enhances bone formation by favoring amino acid import and collagen synthesis in osteoblasts, a function requiring its phosphorylation by RSK2, the kinase inactivated in Coffin-Lowry Syndrome. Here, we show that in contrast, RSK2 activity, ATF4-dependent collagen synthesis, and bone formation are increased in mice lacking neurofibromin in osteoblasts (Nf1(ob)(-/-) mice). Independently of RSK2, ATF4 phosphorylation by PKA is enhanced in Nf1(ob)(-/-) mice, thereby increasing Rankl expression, osteoclast differentiation, and bone resorption. In agreement with ATF4 function in amino acid transport, a low-protein diet decreased bone protein synthesis and normalized bone formation and bone mass in Nf1(ob)(-/-) mice without affecting other organ weight, while a high-protein diet overcame Atf4(-/-) and Rsk2(-/-) mice developmental defects, perinatal lethality, and low bone mass. By showing that ATF4-dependent skeletal dysplasiae are treatable by dietary manipulations, this study reveals a molecular connection between nutrition and skeletal development.

Strontium ranelate: a novel concept for the treatment of osteoporosis

The di-strontium salt strontium ranelate, a novel orally active agent consisting of two atoms of stable strontium and the organic moiety ranelic acid, has been developed for the treatment of osteoporosis. It has been shown to enhance osteoblastic cell replication and increase collagen synthesis while decreasing pre-osteoclast differentiation and bone-resorbing activity of mature osteoclasts in vitro. Studies performed in healthy animals have shown that strontium ranelate not only increases bone mass at various skeletal sites but also improves mechanical properties of femoral, humeral and vertebral bones. In estrogen-deficient animals, strontium ranelate effectively inhibits increased bone resorption while maintaining bone formation. Similar results have been obtained from studies in hind limb-immobilized animals. Strontium ranelate has been investigated in a large phase III program which includes two extensive multinational, randomized, double-blind, placebo-controlled clinical trials for the treatment of severe postmenopausal osteoporosis. Before inclusion in these two studies, patients were subjected to a run-in study in order to initiate normalization of their calcium and vitamin D status. The SOTI study, aimed at assessing strontium ranelate's effect on the risk of vertebral fractures, revealed a significant reduction in the risk of new vertebral fractures in the group treated with strontium ranelate. The TROPOS study, which aimed at evaluating the effect of strontium ranelate on nonvertebral fractures, showed a significant reduction in the risk of new nonvertebral fractures and also, in a high-risk subgroup, a significant reduction in the risk of hip fractures. In both studies, the reduction in fracture risk was accompanied by an increase in bone mineral density. The increase remained significant also after correction of the bone mineral density for the higher atomic mass of strontium compared to calcium. Furthermore, an increase in serum levels of a bone formation marker and a decrease in serum levels of a bone resorption marker were observed. The spectrum of side effects comprised mild diarrhea, particularly within the first weeks of treatment, and also a slightly increased risk of venous thromboembolic events. Strontium ranelate thus appears to be a new, effective treatment to prevent fractures in postmenopausal osteoporosis with a novel mechanism of action. It can be expected for the near future that strontium ranelate will also be evaluated for other types of osteoporosis, such as male osteoporosis.

RANK ligand and TNF-α mediate acid-induced bone calcium efflux in vitro

Chronic metabolic acidosis stimulates net calcium efflux from bone due to increased osteoclastic bone resorption and decreased osteoblastic collagen synthesis. Previously, we determined that incubation of neonatal mouse calvariae in medium simulating physiological metabolic acidosis leads to a significant, cyclooxygenase-dependent, increase in RNA for bone cell receptor activator of NF-kappaB ligand (RANKL) compared with incubation in neutral pH medium. In this study, we tested the hypothesis that the acid-mediated increase in RANKL expression is a primary mechanism for the stimulated osteoclastic resorption. Acid medium increased the medium concentration of sRANKL without altering the concentration of the decoy receptor osteoprotegerin (OPG). Inhibition of the RANKL pathway with concentrations of OPG up to 25 ng/ml, far greater than physiological, did not significantly decrease the robust acid-induced Ca efflux from bone nor did incubation of the calvariae with a different inhibitor, RANK/Fc (up to 50 ng/ml). Thus acid-induced net Ca efflux appears to involve mechanisms in addition to the RANK/RANKL pathway. Osteoblasts also produce TNF-alpha, another cytokine that stimulates the maturation and activity of osteoclasts. Incubation of calvariae in acid medium caused a significant increase in TNF-alpha levels. Incubation of calvariae with anti-TNF (up to 250 ng/ml) did not significantly decrease acid-induced net Ca efflux. However, the combination of RANK/Fc plus anti-TNF caused a significant but subtotal reduction in acid-induced Ca efflux, whereas the combination of RANK/Fc plus an isotype-matched control for the anti-TNF had no effect on Ca release. Thus simultaneous inhibition of RANKL and TNF-alpha is necessary to reduce acid-induced, cell-mediated net Ca efflux from bone; however, additional osteoblast-produced factors must also be involved in acid-induced, cell-mediated bone resorption.

A Randomized Placebo-Controlled Trial of Short-Term Graded Transdermal Estradiol in Healthy Gonadotropin-Releasing Hormone Agonist-Suppressed Pre- and Postmenopausal Women: Effects on Serum Markers of Bone Turnover, Insulin-Like Growth Factor-I, and Osteoclastogenic Mediators

The acute effects of estradiol on procollagen type 1 formation in pre- and postmenopausal women are controversial. Twenty-three premenopausal women and 13 postmenopausal women received two consecutive im injections of 3.75 mg leuprolide acetate 3 wk apart to block endogenous ovarian steroidogenesis. Transdermal estradiol therapy commenced on the night of the second leuprolide injection in all subjects, except five pre- and two postmenopausal women who were randomized to receive placebo patches. Estradiol therapy was applied incrementally, with each dose of 0.05, 0.10, 0.15, and 0.20 mg/d administered for 4 consecutive days, to mimic the estradiol changes typifying the follicular phase of the menstrual cycle. Blood aminoterminal propeptide of type I procollagen (PINP), intact osteocalcin (OC), carboxyterminal telopeptide of type I collagen (CTx), IGF-I, and estradiol were measured before and at the end of each estradiol increment. Potential mediators such as osteoprotegerin and receptor activator of nuclear factor-kappaB ligand (RANKL) were also measured. Despite comparable increases in serum estradiol, PINP increased more in postmenopausal compared with premenopausal women (between-group P = 0.03) and occurred at a time when CTx and OC did not change. CTx and IGF-I changed minimally and inconsistently, whereas OC, RANKL, and osteoprotegerin were stable. Repeated measures linear regression disclosed a significant negative association between increases in estradiol and PINP in premenopausal women (P = 0.0006) only. This suggests that lower dose estradiol should greatly increase PINP. Analogous regressions also showed significant negative relationships between changes in estradiol and RANKL in both pre- (P = 0.04) and postmenopausal (P = 0.002) women. Changes in serum markers of bone formation (PINP or OC) did not correlate with those of IGF-I. We conclude that lower dose estradiol rapidly increases osteoblastic collagen synthesis in women at a time when collagen degradation is stable and that this response differs between pre- and postmenopausal women. The effect of estradiol on bone formation does not appear to be mediated by IGF-I. In contrast, RANKL is likely to mediate the effect of estradiol on osteoclastogenesis.

Secondary Causes of Osteoporosis

Secondary Causes of Osteoporosis