Adipogenesis and osteogenesis, a reciprocal relationship in bone marrow, are complex processes including proliferation of precursor cells, commitment to the specific lineage, and terminal differentiation. Accumulating evidence from in vitro and in vivo studies suggests that melatonin affects terminal differentiation of osteoblasts and adipocytes, but little is known about the effect of melatonin on the process of adipogenesis and osteogenesis, especially adipogenesis. This study was performed to determine the effect of melatonin on adipogenesis and osteogenesis in human mesenchymal stem cells (hMSCs). Cell proliferation assays demonstrated that melatonin had no apparent effect on the proliferation of hMSCs. When melatonin was added to the adipogenic/osteogenic medium, it directly inhibited adipogenesis and simultaneously promoted osteogenesis of hMSCs in a dose-dependent manner. Furthermore, quantitative RT-PCR demonstrated that melatonin significantly suppressed peroxisome proliferator-activated receptor gamma (PPARγ) expression (day 3, 25% decrease; day 6, 47% decrease), but promoted Runx2 expression (day 3, 87% increase; day 6, 56% increase) in the early stages of adipogenesis and osteogenesis of hMSCs. Moreover, melatonin down-regulated several markers of terminal adipocyte differentiation, including leptin (30%), lipoprotein lipase (LPL, 41%), adiponectin (51%), and adipocyte protein 2 (αP2, 45%). Meanwhile, melatonin up-regulated several markers of osteoblast differentiation, including alkaline phosphatase (110%), osteopontin (218%), and osteocalcin (310%). These results suggest that melatonin directly inhibits hMSCs adipogenic differentiation and significantly enhances hMSCs osteogenic differentiation by suppressing PPARγ expression and enhancing Runx2 expression; this provides further evidence for melatonin as an anti-osteoporosis drug.

Spines are dendritic protrusions that receive most of the excitatory input in the brain. Early after the onset of cerebral ischemia dendritic spines in the peri-infarct cortex are replaced by areas of focal swelling, and their re-emergence from these varicosities is associated with neurological recovery after acute ischemic stroke (AIS). Urokinase-type plasminogen activator (uPA) is a serine proteinase that plays a central role in tissue remodeling via binding to the urokinase plasminogen activator receptor (uPAR). We report that cerebral cortical neurons release uPA during the recovery phase from ischemic stroke in vivo or hypoxia in vitro. Although uPA does not have an effect on ischemia-or hypoxia-induced neuronal death, genetic deficiency of uPA (uPA Ϫ/Ϫ ) or uPAR (uPAR) abrogates functional recovery after AIS. Treatment with recombinant uPA after ischemic stroke induces neurological recovery in wild-type and uPA Ϫ/Ϫ but not in uPAR Ϫ/Ϫ mice. Diffusion tensor imaging studies indicate that uPA Ϫ/Ϫ mice have increased water diffusivity and decreased anisotropy associated with impaired dendritic spine recovery and decreased length of distal neurites in the peri-infarct cortex. We found that the excitotoxic injury induces the clustering of uPAR in dendritic varicosities, and that the binding of uPA to uPAR promotes the reorganization of the actin cytoskeleton and re-emergence of dendritic filopodia from uPAR-enriched varicosities. This effect is independent of uPA's proteolytic properties and instead is mediated by Rac-regulated profilin expression and cofilin phosphorylation. Our data indicate that binding of uPA to uPAR promotes dendritic spine recovery and improves functional outcome following AIS.

Intramembranous ossification and endochondral ossification are two ways through which bone formation and fracture healing occur. Accumulating amounts of evidence suggests that melatonin affects osteoblast differentiation, but little is known about the effects of melatonin on the process of chondrogenic differentiation. In this study, the effects of melatonin on human mesenchymal stem cells (MSCs) undergoing chondrogenic differentiation were investigated. Cells were induced along chondrogenic differentiation via high-density micromass culture in chondrogenic medium containing vehicle or 50 nm melatonin. Histological study and quantitative analysis of glycosaminoglycan (GAG) showed induced cartilage tissues to be larger and richer in GAG, collagen type II and collagen type X in the melatonin group than in the untreated controls. Real-time RT-PCR analysis demonstrated that melatonin treatment significantly up-regulated the expression of the genes involved in chondrogenic differentiation, including aggrecan (ACAN), collagen type II (COL2A1), collagen type X (COL10A1), SRY (sex-determining region Y)-box 9 (SOX9), runt-related transcription factor 2 (RUNX2) and the potent inducer of chondrogenic differentiation, bone morphogenetic protein 2 (BMP2). And the expression of melatonin membrane receptors (MT) MT1 and MT2 were detected in the chondrogenic-induced-MSCs by immunofluorescence staining. Luzindole, a melatonin receptor antagonist, was found to partially block the ability of melatonin to increase the size and GAG synthesis of the induced cartilage tissues, as well as to completely reverse the effect of melatonin on the gene expression of ACAN, COL2A1, COL10A1, SOX9 and BMP2 after 7 days of differentiation. These findings demonstrate that melatonin enhances chondrogenic differentiation of human MSCs at least partially through melatonin receptors.

Melatonin promotes bone formation and prevents bone degradation via receptor-dependent or receptor-independent actions. The aim of this study is to encapsulate melatonin into poly (lactic-co-glycolic acid) (PLGA) microspheres (PLGA-MEL-MS) and create a melatonin sustained release system, then to evaluate its effect on the osteogenesis of human mesenchymal stem cells (hMSCs) in vitro. PLGA-MEL-MS were prepared by single emulsion solvent evaporation technique. Scanning electron microscopy demonstrated the incorporation of melatonin did not disturb the conventional generation of PLGA microspheres in size and morphology. In vitro drug release assay showed that PLGA-MEL-MS exhibited a biphasic drug release pattern: a low initial burst release effect with approximately 40% drug release at the first 3 days and a relatively retarded and continuous release with about 85% drug release over the 25 days. Cell proliferation assay demonstrated that PLGA-MEL-MS had no apparent effect on proliferation of human MSCs. In an osteogenesis assay, PLGA-MEL-MS obviously enhanced alkaline phosphatase (ALP) mRNA expression and increased ALP activity compared to that in the control group. Meanwhile, several markers of osteoblast differentiation were also significantly upregulated, including runx2, osteopontin, and osteocalcin. Furthermore, quantificational alizarin red-based assay demonstrated that PLGA-MEL-MS significantly enhanced calcium deposit of hMSCs compared to the controls. Therefore, this simple melatonin sustained release system can control released melatonin to generate a microenvironment with a relatively stable concentration of melatonin for a period of time to support osteogenic differentiation of hMSCs in vitro. This suggests that this system may be used as bone growth stimulator in bone healing in vivo.

BackgroundAlthough pyogenic liver abscess (PPLA) fatalities are decreasing owing to early diagnosis and effective treatments, PPLA-associated complications still exist. The purpose of this study was to analyze the characteristic features of initial presentations and final outcomes of PPLA caused by different pathogens.MethodsThis retrospective study collected and analyzed information regarding initial presentations and final outcomes in patients diagnosed with PPLA at admitted at Changhua Christian Hospital from January 1 to December 31, 2010.ResultsDuring the study period, we analyzed the records of a total of 134 patients with documented PPLA. There were no significant causative pathogen-related differences in symptoms at initial presentation. Compared with the survivor group, patients in the mortality group were characterized by male gender (p < 0.001), malignancy (p < 0.001), respiratory distress (p =0.007), low blood pressure (p = 0.024), jaundice (p = < 0.001), rupture of liver abscess (p < 0.001), endophthalmitis (p = 0.003), and multiple organ failure (p < 0.001). No patients received liver transplantation or were diagnosed with HIV during the study period. According to univariate logistic regression analysis, gender (OR = 1.185, 95% CI: 0.284–11.130, p = 0.006), malignancy (OR = 2.067, 95% CI: 1.174–13.130, p = 0.004), respiratory distress (OR = 1.667, 95% CI: 1.164–14.210, p = 0.006), low blood pressure (OR = 2.167, 95% CI: 2.104–13.150, p = 0.003), jaundice (OR = 1.9, 95% CI: 1.246–3.297, p = 0.008), rupture of liver abscess (OR = 5.167, 95% CI: 2.194–23.150, p = 0.003), endophthalmitis (OR = 2.167, 95% CI: 1.234–13.140, p = 0.005), and multiple organ failure (OR = 3.067, 95% CI: 1.184–15.150, p = 0.001) differed significantly between the mortality and survivor groups.ConclusionAlthough the initial presentations of PPLA caused by different pathogens were similar, there were significant differences in mortality in cases involving: (1) male patients, (2) malignancy, (3) initial respiratory distress, (4) initial low blood pressure, (5) jaundice, (6) rupture of liver abscess, (7) endophthalmitis, , and (8) multiple organ failure. We strongly recommend using a severity score of the disease to determine the risk of mortality for each patient with PPLA. In order to prevent complications and reduce mortality, more attention must be paid to high-risk PPLA patients.