Oxysterols enhance osteoblast differentiation in vitro and bone healing in vivo

Division of Diagnostic and Surgical Sciences, University of California at Los Angeles School of Dentistry, Room 53-068 CHS, 10833 Le Conte Avenue, Los Angeles, California 90095, USA.
Journal of Orthopaedic Research (Impact Factor: 2.99). 11/2007; 25(11):1488-97. DOI: 10.1002/jor.20437
Source: PubMed


Oxysterols, naturally occurring cholesterol oxidation products, can induce osteoblast differentiation. Here, we investigated short-term 22(S)-hydroxycholesterol + 20(S)-hydroxycholesterol (SS) exposure on osteoblastic differentiation of marrow stromal cells. We further explored oxysterol ability to promote bone healing in vivo. Osteogenic differentiation was assessed by alkaline phosphatase (ALP) activity, osteocalcin (OCN) mRNA expression, mineralization, and Runx2 DNA binding activity. To explore the effects of osteogenic oxysterols in vivo, we utilized the critical-sized rat calvarial defect model. Poly(lactic-co-glycolic acid) (PLGA) scaffolds alone or coated with 140 ng (low dose) or 1400 ng (high dose) oxysterol cocktail were implanted into the defects. Rats were sacrificed at 6 weeks and examined by three-dimensional (3D) microcomputed tomography (microCT). Bone volume (BV), total volume (TV), and BV/TV ratio were measured. Culture exposure to SS for 10 min significantly increased ALP activity after 4 days, while 2 h exposure significantly increased mineralization after 14 days. Four-hour SS treatment increased OCN mRNA measured after 8 days and nuclear protein binding to an OSE2 site measured after 4 days. The calvarial defects showed slight bone healing in the control group. However, scaffolds adsorbed with low or high-dose oxysterol cocktail significantly enhanced bone formation. Histologic examination confirmed bone formation in the defect sites grafted with oxysterol-adsorbed scaffolds, compared to mostly fibrous tissue in control sites. Our results suggest that brief exposure to osteogenic oxysterols triggered events leading to osteoblastic cell differentiation and function in vitro and bone formation in vivo. These results identify oxysterols as potential agents in local and systemic enhancement of bone formation.

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    • "The third line of evidence is provided by publications which demonstrated that treatment of murine MSC with specific oxysterols in vitro results in enhanced osteoblast differentiation through the activation of several signaling pathways and that LXRs are partially involved in these processes [25–27,29]. In addition, oxysterols have been reported to promote bone healing in vivo [48]. Of note, the same group has shown that pharmacological LXR activation in murine MSC by T0901317 [25] [30] and GW3965 [30] in vitro decreases the expression of markers of osteogenic differentiation. "
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    ABSTRACT: Liver X receptors (LXRs) are nuclear receptors that play a crucial role in the transcriptional control of lipid metabolism. Pharmacological LXR activation is an attractive concept for the treatment of atherosclerosis. Genetic LXR deficiency in mice has been shown to have an effect on bone turnover and structure and LXR activation is known to influence the osteogenic differentiation of bone marrow stromal cells. Therefore, therapeutic pharmacological LXR activation may have relevant effects on bone. Here, using two synthetic LXR ligands, T0901317 and GW3965, we investigated the effect of LXR activation on murine osteoblasts and the influence of long-term LXR activation on bone in vivo in mice. Short term (48 h) in vitro treatment of primary murine osteoblasts with T0901317 resulted in a dose-dependent decrease of osteocalcin and alkaline phosphatase mRNA and protein. In vivo, a 6-day treatment of C57BL/6J mice with T0901317 led to a 40% reduction of serum osteocalcin concentrations. Long-term (12-week) oral administration of T0901317 or GW3965 influenced the expression of established LXR target genes in liver and intestine, but did not alter trabecular and cortical bone structure or bone turnover as determined by total skeleton radiography, histomorphometric analysis of lumbar vertebral trabecular bone, micro CT analysis of femur cortical bone and biochemical determination of bone formation and resorption markers. We conclude that short-term pharmacological LXR activation has the potential to profoundly influence osteoblast function, but that long-term LXR activation in vivo has no adverse effects on the murine skeleton.
    Bone 02/2011; 48(2):339-46. DOI:10.1016/j.bone.2010.08.018 · 3.97 Impact Factor
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    • "Oxysterols, a large family of 27-carbon oxygenated products of cholesterol present in the circulation and in human and animal tissues,(25) are involved in various biologic and pathologic processes, including cholesterol efflux, lipoprotein metabolism, cell differentiation, atherosclerosis, and apoptosis.(26–29) We have demonstrated previously that specific oxysterols stimulate the osteogenic differentiation of pluripotent MSCs and inhibit their adipogenic differentiation through the activation of Hedgehog signaling in vitro(30–33) and enhance bone healing in rat critical-sized calvarial defects in vivo.(34) Here, we report that osteogenic oxysterols are novel activators of expression of the Notch target genes HES-1, HEY-1, and HEY-2 in MSCs. "
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    ABSTRACT: We previously reported that specific oxysterols stimulate osteogenic differentiation of pluripotent bone marrow stromal cells (MSCs) through activation of hedgehog (Hh) signaling and may serve as potential future therapies for intervention in osteopenia and osteoporosis. In this study we report that the osteogenic oxysterol 20(S)-hydroxycholesterol (20S) induces the expression of genes associated with Notch signaling. Using M2-10B4 (M2) MSCs, we found that 20S significantly induced HES-1, HEY-1, and HEY-2 mRNA expression compared with untreated cells, with maximal induction after 48 hours, whereas the nonosteogenic oxysterols did not. Similar observations were made when M2 cells were treated with sonic hedgehog (Shh), and the specific Hh pathway inhibitor cyclopamine blocked 20S-induced Notch target gene expression. 20S did not induce Notch target genes in Smo(-/-) mouse embryonic fibroblasts, further confirming the role of Hh signaling in 20S-induced expression of Notch target genes. Despite the inability of liver X-receptor (LXR) synthetic ligand TO901317 to induce Notch target genes in M2 cells, LXR knockdown studies using siRNA showed inhibition of 20S-induced HEY-1 but not HES-1 expression, suggesting the partial role of LXR signaling in MSC responses to 20S. Moreover, 20S-induced Notch target gene expression was independent of canonical Notch signaling because neither 20S nor Shh induced CBF1 luciferase reporter activity or NICD protein accumulation in the nucleus, which are hallmarks of canonical Notch signaling activation. Finally, HES-1 and HEY-1 siRNA transfection significantly inhibited 20S-induced osteogenic genes, suggesting that the pro-osteogenic effects of 20S are regulated in part by HES-1 and HEY-1.
    Journal of bone and mineral research: the official journal of the American Society for Bone and Mineral Research 10/2009; 25(4):782-95. DOI:10.1359/jbmr.091024 · 6.83 Impact Factor
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    ABSTRACT: This study investigates the distribution of Al and OH defects in synthetic quartz grown from cylindrical seeds parallel to X, Y and Z crystallographic axes. Prismatic habit crystals were grown with m-, R-, r- and x- faces. The growth sectors were identified by radiation-induced coloration and their morphologies were characterized by X-ray topography. Optical and infrared spectroscopies were adopted to evaluate the occurrence of Al-hole, Al-OH and as-grown OH centers. It was found that the relative content of Al-hole and Al-OH centers were dependent on the growth sector and the orientation of the seed crystal. The ratio of Al-hole to Al-OH centers between +X- and -X-sectors grown from the Z-axis seed-crystal is opposite to that found in cylindrical Y-axis and standard Y-bar synthetic quartz crystals. The incorporation of Al-Li centers was responsible for the intense lattice strains observed in r-growth sectors appearing around the X-axis.
    Frequency Control Symposium and Exposition, 2004. Proceedings of the 2004 IEEE International; 09/2004
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