Bone Formation In Cultures Research Articles

Antidiabetic actions of a non-agonist PPARγ ligand blocking Cdk5-mediated phosphorylation

PPARγ is the functioning receptor for the thiazolidinedione (TZD) class of anti-diabetes drugs including rosiglitazone and pioglitazone1. These drugs are full classical agonists for this nuclear receptor, but recent data has shown that many PPARγ-based drugs have a separate biochemical activity, blocking the obesity-linked phosphorylation of PPARγ by Cdk52. Here we describe novel synthetic compounds that have a unique mode of binding to PPARγ, completely lack classical transcriptional agonism and block the Cdk5-mediated phosphorylation in cultured adipocytes and in insulin-resistant mice. Moreover, one such compound, SR1664, has potent anti-diabetic activity while not causing the fluid retention and weight gain that are serious side effects of many of the PPARγ drugs. Unlike TZDs, SR1664 also does not interfere with bone formation in culture. These data illustrate that new classes of anti-diabetes drugs can be developed by specifically targeting the Cdk5-mediated phosphorylation of PPARγ.

Polyphenol Extract of Greens+™ Nutritional Supplement Stimulates Bone Formation in Cultures of Human Osteoblast-like SaOS-2 Cells

Background and Aims: Oxidative stress has been associated with osteoporosis. Greens+™ is a commercially available nutritional supplement containing antioxidative polyphenols. Methods: To study the effects of greens+™ on differentiation and bone formation in human osteoblasts, the cells were cultured in Ham's F-12 medium in the absence or presence of varying concentrations of total free polyphenolic (TFP) in the extracts of greens+™. Results: Our results showed that the number of osteoblasts increased (p <. 05) compared to vehicle control after 2 and 4 days of treatment but were reduced (p <. 05) after 7 days of treatment with 1.2–2.0 mg greens+™ extract/ml (corresponding to 16.8–27.9 ng TFP expressed as gallic acid equivalent per millilter). Lower concentrations of greens+™ extract stimulated alkaline phosphatase activity at early time points (days 9 and 11), while higher concentrations at the later time point of day 13 resulted in a significant (p <. 05) inhibition, in a time (p <. 0001) and dose dependent (p <. 0001) manner. Greens+™ extract stimulated (p <. 05) the mineralized bone nodule formation in a dose and time dependent manner. Conclusions: The results showed that greens+™ extract influenced the maturation of osteoprogenitors toward progression to a bone-forming stage. Our data suggest that greens+™ may have beneficial effects on bone formation in vitro due to its antioxidant polyphenolic content and we can speculate that it may be a good alternative to drugs for the prevention of osteoporosis.

The Effect of Simulated Microgravity by Three-Dimensional Clinostat on Bone Tissue Engineering

Evidence suggests that mechanical stress, including gravity, is associated with osteoblast differentiation and function. To examine effects of microgravity on bone tissue engineering, we used a three-dimensional (3D) clinostat manufactured by Mitsubishi Heavy Industries (Kobe, Japan). A 3D clinostat is a device that generates multidirectional G force. By controlled rotation on two axes, it cancels the cumulative gravity vector at the center of the device. We cultured rat marrow mesenchymal cells (MMCs) in the pores of interconnected porous calcium hydroxyapatite (IP-CHA) for 2 weeks in the presence of dexamethasone using the 3D clinostat (clinostat group). MMCs cultured using the 3D clinostat exhibited a 40% decrease in alkaline phosphatase activity (a marker of osteoblastic differentiation), compared with control static cultures (control group). SEM analysis revealed that although there was no difference between the two groups in number or distribution of cells in the pores, the clinostat group exhibited less extensive extracellular matrix formation than the control group. Cultured IP-CHA/MMC composites were then implanted into subcutaneous sites of syngeneic rats and harvested 8 weeks after implantation. All implants showed bone formation inside the pores, as indicated by decalcified histological sections and microfocus computed tomography. However, the volume of newly formed bone was significantly lower for the clinostat group than for the control group, especially in the superficial pores close to the implant surface. These results indicate that new bone formation in culture was inhibited by use of the 3D clinostat, and that this inhibition was mainly due to suppression of osteoblastic differentiation of MMCs.

Synthesis of the Blood Circulating C-terminal Fragment of Insulin-like Growth Factor (IGF)-binding Protein-4 in Its Native Conformation: CRYSTALLIZATION, HEPARIN AND IGF BINDING, AND OSTEOGENIC ACTIVITY

Insulin-like growth factor-binding proteins play a critical role in a wide variety of important physiological processes. It has been demonstrated that both an N-terminal and a C-terminal fragment of insulin-like growth factor-binding protein-4 exist and accumulate in the circulatory system, these fragments accounting for virtually the whole amino acid sequence of the protein. The circulating C-terminal fragment establishes three disulfide bridges, and the binding pattern of these has recently been defined. Here we show that the monodimensional 1H NMR spectrum of the C-terminal fragment is typical of a protein with a relatively close packed tertiary structure. This fragment can be produced in its native conformation in Escherichia coli, without the requirement of further refolding procedures, when synthesis is coupled to its secretion from the cell. The recombinant protein crystallizes with the unit cell parameters of a hexagonal system. Furthermore, it binds strongly to heparin, acquiring a well defined oligomeric structure that interacts with insulin-like growth factors, and promotes bone formation in cultures of murine calvariae.

Effects of Lactobacillus helveticus fermented milk on bone cells in vitro

Milk fermented with Lactobacillus helveticus ( L. helveticus) contains small peptides such as isoleucyl-prolyl-proline (IPP) and valyl-prolyl-proline (VPP), which inhibit the angiotensin converting enzyme (ACE). We investigated the effects of L. helveticus fermented milk whey ( Lh-whey) and its components, sour milk whey, calcium and IPP and VPP peptides, on bone cells in vitro. An osteoblast assay was performed by determining the amount of deposited calcium as an index of bone formation in cultures of mouse osteoblasts formed from bone marrow-derived osteoblast precursor cells. An osteoclast assay was performed by determining the activity of tartrate-resistant acid phosphatase released into the culture medium in cultures of mouse osteoclasts formed from bone marrow-derived osteoclast precursor cells. The Lh-whey increased bone formation 1.3–1.4 times with the 1 × 10 −5, 1 × 10 −4 and 1 × 10 −3 solutions. The IPP and VPP peptides also demonstrated a significant 5-fold activation of bone formation in in vitro osteoblast cultures, whereas the sour milk whey and calcium had no effect. No significant effects were observed on osteoclasts in vitro with any of the study products. L. helveticus fermented milk whey contains bioactive components that increase osteoblastic bone formation in vitro. The effect may be due to the ACE-inhibitory IPP and VPP peptides, which showed a similar effect to that of the L. helveticus fermented milk whey.

Mechanisms of Osteoblastic Metastases: Role of Endothelin-1

Certain solid tumors metastasize to bone, causing an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not understood completely. We identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provide evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. Tumor-conditioned media and exogenous ET-1 stimulated osteoblast proliferation and new bone formation in cultures of calvarias from mice. These effects were blocked by endothelin A (ETA) but not by ETB receptor antagonists. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ETA receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on cell growth in vitro or at the orthotopic site (mammary fat pad) of ZR-75-1, or MDA-MB-231 cells. Collectively, the data suggest that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. Endothelin A receptor blockade may be useful for the prevention and treatment of osteoblastic bone metastases attributable to breast or prostate cancer.

Role of endothelin-1 in osteoblastic bone metastases.

Certain solid tumors metastasize to bone and cause an osteoblastic response. The mechanisms by which tumor cells stimulate this new bone formation are not completely understood. The authors identified three breast cancer lines that cause osteoblastic metastases in female nude mice and provided evidence that tumor-produced endothelin-1 (ET-1) mediates the osteoblastic response. Tumor conditioned media, as well as exogenous ET-1, stimulated osteoblast proliferation and new bone formation in cultures of mouse calvariae. These effects were blocked by antagonists of the endothelin A (ET(A)), but not ET(B), receptors. Mice inoculated with the ZR-75-1 breast cancer line and treated with a selective ET(A) receptor antagonist (ABT-627) had significantly fewer osteoblastic bone metastases and less tumor burden compared with untreated mice. In contrast, there was no effect of ABT-627 on osteolytic bone metastases caused by ET-1-negative breast cancer, MDA-MB-231. ABT-627 had no effect on growth in vitro or at the orthotopic site of ZR-75-1 or MDA-MB-231 cells. Collectively, the data suggested that tumor-produced ET-1 mediates osteoblastic bone metastases by stimulating osteoblast proliferation and new bone formation. ET(A) receptor blockade may be useful for prevention and the treatment of osteoblastic bone metastases due to breast or prostate cancer.

Effects of deflazacort on aspects of bone formation in cultures of intact calvariae and osteoblast-enriched cells

Deflazacort, a synthetic glucocorticoid reported to have bone-sparing properties in vivo, and cortisol were compared for their effects on bone formation in vitro. Deflazacort and cortisol were studied for their effects on DNA and collagen synthesis in cultures of intact fetal rat calvariae and of osteoblast-enriched (Ob) cells from 21- to 22-day-old fetal rat parietal bone. Both steroids were also examined for their effects on skeletal insulin-like growth factor (IGF) I production, which is decreased by cortisol and appears relevant to its mode of action. After 24 h of culture, deflazacort and cortisol had limited effects on the parameters studied, although cortisol at 100 nM decreased [3H]proline incorporation into collagen in intact calvariae. In contrast, after 72 h deflazacort and cortisol at 1-100 nM inhibited the incorporation of [3H]thymidine into DNA and at 100 nM decreased the incorporation of [3H]proline into collagen and noncollagen protein in intact calvariae. Deflazacort and cortisol at 10-1000 nM decreased calvarial collagen degradation to a similar extent. Both steroids had a similar activity, and at 100 nM for 72 h they decreased IGF-I production by calvariae; however, cortisol at 10 nM was somewhat more effective than deflazacort in decreasing IGF-I levels. Deflazacort and cortisol had analogous effects in Ob cell cultures. After 24 h of treatment, deflazacort at 100-1000 nM and cortisol at 10-1000 nM decreased the labeling of DNA, and both steroids at 100-1000 nM caused a similar decrease in [3H]proline incorporation into collagen in Ob cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of bone nodule formation in vitro by prostaglandins E1 and E2.

It has been established by organ culture experiments that prostaglandins (PGs) stimulate bone resorption in vitro. Experiments in vivo and with organ cultures suggest that PGs may also stimulate bone formation, and that bone formation in response to a variety of environmental stimuli is PG dependent. We have tested the ability of PGE1, PGE2, and PGF2 alpha to induce bone formation in cultures of rat calvarial cells. PGE1 and PGE2 significantly increased bone nodule formation at concentrations of 10(-8) M and above, to reach 3 times the control levels at 10(-7) M. PGF2 alpha was without effect. The increase in the number of nodules was effected without a significant change in the number of cells in control and test cultures with a logarithmic phase of growth, and there was no increase in the average size of the nodules. This suggests that PGs acted through induction of nodule formation by a population of cells that would not otherwise have produced nodules. Nodules were induced by PGs if the PGs were present in the early stages of the cultures; osteoblastic cells incubated with PGs for 8 days produced very similar numbers of nodules as cultures incubated with PGs throughout the 21-day culture period, although nodules did not become identifiable until 8-10 days of incubation. The addition of PGs late in the culture period had little effect on nodule formation. These experiments identify a role for PGs in bone formation in vitro, which may represent a pathway common to the bone anabolism that is observed in response to many environmental stimuli.

Stimulation of bone nodule formation in vitro by prostaglandins E1 and E2

It has been established by organ culture experiments that prostaglandins (PGs) stimulate bone resorption in vitro. Experiments in vivo and with organ cultures suggest that PGs may also stimulate bone formation, and that bone formation in response to a variety of environmental stimuli is PG dependent. We have tested the ability of PGE1, PGE2, and PGF2 alpha to induce bone formation in cultures of rat calvarial cells. PGE1 and PGE2 significantly increased bone nodule formation at concentrations of 10(-8) M and above, to reach 3 times the control levels at 10(-7) M. PGF2 alpha was without effect. The increase in the number of nodules was effected without a significant change in the number of cells in control and test cultures with a logarithmic phase of growth, and there was no increase in the average size of the nodules. This suggests that PGs acted through induction of nodule formation by a population of cells that would not otherwise have produced nodules. Nodules were induced by PGs if the PGs were present in the early stages of the cultures; osteoblastic cells incubated with PGs for 8 days produced very similar numbers of nodules as cultures incubated with PGs throughout the 21-day culture period, although nodules did not become identifiable until 8-10 days of incubation. The addition of PGs late in the culture period had little effect on nodule formation. These experiments identify a role for PGs in bone formation in vitro, which may represent a pathway common to the bone anabolism that is observed in response to many environmental stimuli.

Differential Effects of Continuous and Transient Treatment with Parathyroid Hormone Related Peptide (PTHrp) on Bone Collagen Synthesis*

Parathyroid hormone-related peptide (PTHrp), a polypeptide synthesized by tumors associated with hypercalcemia and known to cause bone resorption, was examined for its effects on bone formation in cultures of 21-day fetal rat calvariae. Continuous treatment with PTHrp for 24-72 h stimulated DNA synthesis, but inhibited [3H] proline incorporation into collagen by about 50%. In contrast, transient exposure to PTHrp at 0.1-1.0 nM for 24 h followed by removal of the factor for 48 h caused an increase in [3H]proline incorporation into collagen and noncollagen protein by 2- and 1.6-fold, respectively. The stimulatory effect was seen in the periosteum-free bone, and was decreased, but not prevented by hydroxyurea. PTHrp at 1-10 nM for 24 h increased medium insulin-like growth factor (IGF) I levels by 2.5-4.4-fold, and the effect was sustained 48 h after the removal of the agent. An IGF I neutralizing antibody prevented the stimulatory effect of PTHrp on bone collagen synthesis. PTH had the same stimulatory effects as those of PTHrp on bone collagen synthesis and IGF I concentrations, although slightly lower doses were needed to observe the enhancement of [3H]proline incorporation into collagen. It is concluded that continuous treatment with PTHrp inhibits, whereas transient treatment stimulates, collagen synthesis; the stimulatory effect appears mediated by an enhancement in the local production of IGF I.

Isolation and characterization of insulin-like growth factor I (somatomedin-C) from cultures of fetal rat calvariae.

Cultured bones have been shown to secrete local regulators of bone remodeling, such as beta 2-microglobulin, transforming growth factor-beta, and insulin-like growth factor (IGF), but the IGF secreted has not been characterized. In the present study, IGF from medium conditioned by 21-day-old fetal rat calvariae was isolated and characterized. IGF was purified using dialysis, gel filtration, and reverse phase HPLC. Amino acid composition was compatible with that of IGF I (somatomedin-C), and amino-terminal sequence analysis revealed homology with IGF-I. The concentration of IGF-I in the calvarial culture medium was 1 nM and was suppressed by cycloheximide. Calvaria-derived rat IGF I at 20 nM stimulated DNA and collagen synthesis by 42% and 26%, respectively, in monolayer cultures of osteoblast-rich rat parietal bone cells. This study indicates that locally produced IGF-I regulates bone formation in cultures of 21-day-old fetal rat calvariae.

Interleukin-1 has independent effects on deoxyribonucleic acid and collagen synthesis in cultures of rat calvariae.

Interleukin-1 (IL-1), a monokine known to be important in host defense mechanisms and recently reported to stimulate bone resorption, was studied for its effects on bone formation in cultures of 21-day-old fetal rat calvariae. IL-1 at 0.1-5 U/ml stimulated the incorporation of [3H] thymidine into acid-insoluble residues (DNA) by 29-123% in calvariae treated for 24-96 h. IL-1 also increased the bone DNA content and the number of mitoses after colcemid arrest. IL-1 stimulated total protein synthesis. Treatment with IL-1 at 0.01-1 U/ml for 24 h caused a small increase in the incorporation of [3H]proline into collagenase-digestible protein (CDP) and non-collagen protein (NCP). However, higher doses of IL-1 (5 U/ml) or longer exposure to the agent (1 U/ml for 96 h) inhibited the labeling of CDP but not of NCP. IL-1 affected only type I collagen. The stimulatory effects of IL-1 on DNA, CDP, and NCP labeling were independent, since they were observed at different doses, and hydroxyurea abolished the effect on DNA without changing that on CDP and NCP labeling. Indomethacin blocked the stimulatory effect on CDP and NCP labeling, suggesting a prostaglandin-mediated effect, but did not change the IL-1 effect on DNA synthesis. These studies indicate that IL-1 stimulates calvarial DNA, collagen, and NCP synthesis, but exposure of the calvariae to high IL-1 doses or to IL-1 for prolonged periods of time results in an inhibition of collagen synthesis.

Effect of sodium vanadate on deoxyribonucleic acid and protein syntheses in cultured rat calvariae.

Sodium vanadate, an agent known to have multiple cellular actions, was studied for its effects on aspects of bone formation in cultures of 21-day-old fetal rat calvariae. Vanadate (0.1-10 microM) stimulated the incorporation of [3H] thymidine into acid-insoluble residues (DNA); the effect appeared after 3 h and was sustained for 96 h. Vanadate increased the bone DNA content and mitotic index. Treatment with vanadate at 10 microM for 24 h or at 0.3-1 microM for 96 h increased the incorporation of [3H]proline into collagenase-digestible protein (CDP), but the effect was not specific for collagen; vanadate also increased the labeling of noncollagen protein (NCP). Vanadate increased the incorporation of [3H]proline into type I collagen without affecting other collagen types. Vanadate (100 microM) caused a marked and irreversible inhibitory effect on the labeling of DNA, CDP, and NCP. Treatment with vanadate at multiple doses for 3-96 h did not stimulate alkaline phosphatase activity, but this enzyme was inhibited in bones exposed to 1 mM vanadate for 24 h or 10 microM vanadate for 96 h. The stimulatory effect on DNA labeling was primarily observed in the periosteum, while that on CDP labeling was seen only in the periosteum-free bone. These studies indicate that sodium vanadate stimulates bone DNA, collagen, and NCP syntheses in vitro, although high doses of vanadate have an irreversible inhibitory effect.

The fate of rat bone marrow, spleen and periosteum cultivated in vivo in the diffusion chamber, with special reference to bone formation

The histogenesis of bone formation in cultures in vivo of bone marrow and of periosteum is described. Bone formation was observed in 59 per cent of explants of rat bone marrow cultivated in diffusion chambers in the abdomen of the rat for three weeks or more. In explanted periosteum from the rat femur, bone formation was seen in 33.5 per cent of the explants. No bone formation was found in explanted rat spleen containing hematopoietic foci. It is evident that under the described conditions bone formation occurred only in the presence of osteogenic cells. It cannot be decided, on the basis of these experiments, whether bone formation in the bone marrow cultures took place by the growth of surviving osteogenic cells or by the humoral influence of necrotizing bone splinters on the reticulum cells of the bone marrow.