Hui Sheng

Durable Mesenchymal Stem Cell Labelling by Using Polyhedral Superparamagnetic Iron Oxide Nanoparticles

Small polyhedral superparamagnetic iron oxide (SPIO) nanoparticles (<10 nm) coated with a thin layer of silica were prepared (SPIO@SiO(2) and SPIO@SiO(2)-NH(2)). Surface modification of the small polyhedral silica-coated SPIO nanoparticles with amines led to substantially higher mesenchymal stem cell (MSC) labelling efficiency without the use of additional transfecting agents. Therefore, amine surface-modified nanoparticles (SPIO@ SiO(2)-NH(2)) appeared to be the preferred candidate for MSC labelling. In vitro studies demonstrated that controlled labelling of SPIO@SiO(2) and SPIO@SiO(2)-NH(2) did not cause MSC death or proliferation inhibition. MSCs labelled with SPIO@SiO(2)-NH(2) nanoparticles retained differentiation potential and showed osteogenic, adipogenic and chondrogenic differentiations. The noncytotoxic polyhedral SPIO@SiO(2)-NH(2) nanoparticle-labelled MSCs were successfully implanted in rabbit brain and erector spinae muscle, and demonstrated long-lasting, durable MRI labelling efficacy after 8-12 weeks.

Continuous Occurrence of Both Insufficient Neovascularization and Elevated Vascular Permeability in Rabbit Proximal Femur During Inadequate Repair of Steroid-Associated Osteonecrotic Lesions

OBJECTIVE To examine the features of the intraosseous vasculature, the size of the marrow stem cell pool (MSCP), and expression of vascular endothelial growth factor A (VEGF) during inadequate repair of steroid-associated osteonecrotic lesions in rabbits. METHODS Steroid-associated osteonecrosis was induced in male rabbits. At 0, 1, 2, 4, and 6 weeks postinduction, vascularization and permeability indices were quantified by dynamic magnetic resonance imaging (MRI). In addition, the size of the MSCP in the hematopoietic and mesenchymal compartments was determined, and marrow mononuclear cells expressing specific surface markers for endothelial progenitor cells or periendothelial mural precursor cells were counted. At various time points after the rabbits were killed, the proximal femora were dissected to examine the intraosseous vasculature by angiography, histomorphometry, and ultramorphology. In addition, osteonecrotic lesion repair and marrow VEGF expression were evaluated. RESULTS Lesion formation without repair was observed at 2 weeks after induction of steroid-associated osteonecrosis. Rabbits displaying destructive repair (DR+) and those displaying reparative osteogenesis (DR-) from 4 weeks to 6 weeks postinduction were identified. From week 2 to week 6, the vascularization index was significantly lower in DR+ rabbits compared with DR- rabbits, whereas the permeability index was significantly higher in DR+ rabbits compared with DR- rabbits. The features of the intraosseous vasculature determined by angiography, histomorphometry, and ultramorphology were consistent with those determined by dynamic MRI. The MSCP size and number of marrow mononuclear cells expressing specific surface markers were all significantly lower in DR+ rabbits than in DR- rabbits from week 1 to week 6. The increased VEGF expression at 2 weeks was maintained through week 6 in DR+ rabbits, whereas VEGF expression decreased in DR- rabbits from week 2 to week 6. CONCLUSION Continuous occurrence of both insufficient neovascularization and elevated vascular permeability is accompanied by a continuously low- level MSCP and uncontrolled VEGF expression during inadequate repair of steroid-associated osteonecrotic lesions.

Association between metabolic syndrome and osteoporosis: A meta-analysis

Metabolic syndrome (MetS) consists of several independent clinically recognisable features that have their own independent effects on bone metabolism, and even a single disease could have apparently contradictory effects on bone metabolism. This meta-analysis aimed to detect a relationship between MetS and osteoporosis. The PubMed, Embase, and Cochrane library databases were searched for relevant studies published before April 1, 2013. On June 22, 2013, the databases were searched again for additional studies. Studies clearly reporting a comparison of bone mineral density (BMD) in subjects with or without MetS were selected for our analysis. From these results, we analysed the crude BMD and the BMD adjusted for all covariates (age, weight, height, alcohol intake, cigarette smoking and exercise). Weighted mean differences were calculated using a random-effects model. Nine studies were included in this meta-analysis. No significant differences were found when analysing the crude lumbar spine BMD and the femoral neck BMD. However, the MetS (-) group showed a significantly higher BMD when all covariates were adjusted for femoral neck BMD (0.02, p=0.0002) and lumbar spine BMD (0.01, p=0.007). Subgroup analysis suggested a negative effect of MetS on BMD in men but not in women. These findings suggest that MetS is a risk factor for developing osteoporosis in men.

Constitutional Flavonoids Derived from Epimedium Dose-Dependently Reduce Incidence of Steroid-Associated Osteonecrosis Not via Direct Action by Themselves on Potential Cellular Targets

Intravascular-thrombosis and extravascular-lipid-deposit are the two key pathogenic events considered to interrupt intraosseous blood supply during development of steroid-associated osteonecrosis (ON). However, there are no clinically employed agents capable of simultaneously targeting these two key pathogenic events. The present experimental study demonstrated that constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common stem nuclear) exerted dose-dependent effect on inhibition of both thrombosis and lipid-deposition and accordingly reducing incidence of steroid-associated ON in rabbits, which was not via direct action by themselves rather by their common metabolite on potential cellular targets involved in the two pathogenic pathways. The underlying mechanism could be explained by counteracting endothelium injury and excessive adipogenesis. These findings encourage designing clinical trials to investigate potential of EF in prevention of steroid-associated ON.

A Novel Semisynthetic Molecule Icaritin Stimulates Osteogenic Differentiation and Inhibits Adipogenesis of Mesenchymal Stem Cells

Background We previously reported that the constitutional flavonoid glycosides derived from herb Epimedium (EF, composed of seven flavonoid compounds with common nuclear stem) exerted beneficial effects on the bone, including promoting bone formation and inhibiting bone marrow fat deposition. Recent in vivo study showed that Icaritin was a common metabolite of these constitutional flavonoid glycosides, indicating that Icaritin is a bioactive compound. The present study was designed to investigate whether Icaritin could promote osteogenic differentiation and suppress adipogenic differentiation of marrow mesenchymal stem cells (MSCs). Methods Primary MSCs were harvested from adult mice and exposed to Icaritin to evaluate whether it could promote osteogenesis and suppress adipogenesis using the following assays: determination of alkaline phosphatase (ALP) activity and mineralization; mRNA expression of osteogenic differentiation marker Runx2; osteocalcin and bone sialoprotein (BSP) by RT-PCR; quantification of adipocyte-like cells by Oil Red O staining assay and mRNA expression for adipogenic differentiation markers peroxisome proliferator-activated receptor gamma (PPARγ); adipocyte fatty acid binding protein (aP2) and lipoprotein lipase (LPL) by RT-PCR. For the underlying mechanism, glycogen synthase kinase-3beta (GSK3β) and β-catenin were also explored by western blotting. Results Icaritin promoted osteogenic differentiation and maturation of MSCs as indicated by increased mRNA expression for Runx2, osteocalcin and BSP, and enhanced ALP activity and mineralization; Icaritin inhibited adipogenic differentiation, as indicated by decreased mRNA expression for PPARγ, LPL, aP2, and suppressed formation of adipocyte-like cells; Icaritin inactivated GSK3β and suppressed PPARγ expression when promoting osteogenesis and suppressing adipogenesis of MSCs. Conclusion This was the first study demonstrating that the novel semisynthetic molecule Icaritin could stimulate osteogenic differentiation and inhibit adipogenesis of MSCs, which was associated with the suppression of GSK3β and PPARγ.

Promotion of bone repair by implantation of cryopreserved bone marrow–derived mononuclear cells in a rabbit model of steroid‐associated osteonecrosis

OBJECTIVE Cytotherapy is an insufficient method for promoting bone repair in steroid-associated osteonecrosis (SAON), and this has been attributed to impairment of the bioactivity of bone marrow-derived stem cells (BMSCs) after pulsed administration of steroids. Cryopreserved autologous bone marrow-derived mononuclear cells (BMMNCs), which contain BMSCs, might maintain their bioactivity in vitro. This study sought to investigate the effects of cryopreserved BMMNCs, before steroid administration, on the enhancement of bone repair in an established rabbit model of SAON. METHODS For in vitro study, bone marrow was harvested 4 weeks before SAON induction from the iliac crests of rabbits (n = 10) to isolate fresh BMMNCs, and the BMMNCs were then cryopreserved for 8 weeks. Both the fresh and the cryopreserved BMMNCs were evaluated for their bioactivity and osteogenic differentiation capacity. In addition, BMMNCs were isolated 2 weeks after SAON induction and subjected to the same evaluations. For in vivo study, cryopreserved BMMNCs were implanted into the bone tunnel during core decompression of the femur (n = 12 rabbits) after the induction of SAON, and tissue regeneration was evaluated by micro-computed tomography and histologic analyses at 12 weeks postoperation. RESULTS In vitro, there were no significant differences in the bioactivity or ability to undergo osteogenic differentiation between fresh BMMNCs and cryopreserved BMMNCs, but after SAON induction, both features were decreased significantly. In vivo, the bone mineral density, ratio of bone volume to total volume of bone, and volume and diameter of neovascularization within the bone tunnel were significantly higher in the BMMNC-treated group compared to the nontreated control group at 12 weeks postoperation. CONCLUSION Cryopreserved BMMNCs maintained their bioactivity and promoted bone regeneration and neovascularization within the bone tunnel after core decompression in this rabbit model of SAON.

Differential pattern for regulating insulin secretion, insulin resistance, and lipid metabolism by osteocalcin in male and female T2DM patients

Background Osteocalcin has been reported to be relevant to glucose and lipid metabolism, indicating it may stimulate insulin secretion and improve insulin resistance. Yet the difference between male and female patients is still not clear. We aimed to investigate the difference in serum osteocalcin, and its association with glucose, lipid metabolism, pancreatic function, insulin sensitivity, and resistance in male and female middle-aged and elderly type 2 diabetic (T2DM) patients. Material/Methods 739 T2DM patients were included. After measurement of body mass index (BMI), the levels of fasting plasma glucose (FPG), insulin (FINS), C peptide (FC-P), 2-h post-OGTT plasma glucose (2h-PG), HbA1C, and osteocalcin were determined. Homeostasis model assessment of β-cell function (HOMA-%B), homeostasis model assessment of insulin sensitivity (HOMA-%S), and homeostasis model assessment of insulin resistance (HOMA-IR) were calculated. Results Females had higher osteocalcin concentration than males (P<0.05). In males, serum osteocalcin was negatively correlated with HbA1C, FPG, and 2-h PG (P<0.05), but positively with 2-h post-OGTT C peptide (2hC-P), 2-h post-OGTT serum insulin (2h-INS), and HOMA-%B (P<0.05). In females, serum osteocalcin was negatively correlated with HbA1C, FPG, triglyceride (TG), and HOMA-IR (P<0.05), but positively with 2-h C-P, 2-h INS, HOMA-%B, HOMA-%S, and high-density lipoprotein (HDL) (P<0.05). In all subjects, serum osteocalcin was inversely correlated with HbA1C, FPG, and 2-h PG (P<0.05), but positively with 2-h C-P, 2-h INS, HDL, and HOMA-%B (P<0.05). Conclusions Osteocalcin might improve glucose metabolism through enhancing insulin secretion in males, and through increasing insulin secretion and improving insulin resistance in females with T2DM. Osteocalcin probably also plays an important role in lipid metabolism.

Low bone mineral density in chinese adults with nonalcoholic Fatty liver disease.

Aim. To investigate bone metabolic characteristics in Chinese adults with nonalcoholic fatty liver disease (NAFLD). Methods. A total of 224 patients (99 males and 125 postmenopausal females) were recruited and divided into 4 groups: males without NAFLD, males with NAFLD, females without NAFLD, and females with NAFLD. Bone mineral density (BMD) was evaluated according to body mass index (BMI), waist circumference (WC), and serum biomarkers. β cell function was evaluated by HOMA2%B, HOMA2%S, and HOMA2IR. Results. Males in the NAFLD group had lower BMD of the right hip and the femoral neck (0.852 ± 0.117 versus 0.930 ± 0.123, P = 0.002; 0.736 ± 0.119 versus 0.812 ± 0.132, P = 0.004), and females had lower BMD of the right hip (0.725 ± 0.141 versus 0.805 ± 0.145, P = 0.002) even after adjusted for weight, BMI, waist, HDL, and ALT. There was no significant difference in bone metabolic markers between patients with and without NAFLD. NAFLD was an important factor that affected the bone; moreover, the effect attenuated when HOMA2IR entered into the model (R 2 = 0.160, β = −0.172, and P = 0.008). Conclusions. NAFLD exerts a detrimental effect on BMD in both males and females. Insulin resistance may play an important role in this pathophysiological process.

Functional perfusion MRI predicts later occurrence of steroid‐associated osteonecrosis: An experimental study in rabbits

Ischemia is the defined pathway leading to steroid‐associated osteonecrosis (ON). Early detection of ischemic condition may help predict later ON occurrence. Bone marrow perfusion function evaluation by perfusion magnetic resonance imaging (MRI) may be a unique modality for this application. Twenty‐five adult male New Zealand white rabbits were used in this study. Lipopolysaccharide (LPS) and methylprednisolone (MPS) were administrated for ON induction based on a published protocol. T1‐weighted and fat suppression T2‐weighted MR imaging (conventional MRI) were performed for ON lesion detection based on the abnormal signal in the proximal femora at week 0 as the baseline (before LPS injection), and week 1 and week 2 after MPS injection. At the same time, the blood perfusion function in the proximal femora was measured by perfusion MRI. Maximum enhancement (ME)—an index of MRI perfusion function was analyzed. After MRI scanning, the proximal femora were prepared histopathologically for ON lesion analysis. The rabbit with bilateral histopathological ON lesions was defined as an ON+ rabbit and included in the ON+ group evaluated at week 1 and week 2, respectively, and the rabbit without ON lesions in bilateral femora was classified into the ON− group. For the underlying mechanism of perfusion change, the extravascular marrow fat cells were measured and the intravascular endothelium inflammation injury indicator of tissue factor (TF) expression and thrombus formation were detected. In ON+ group, ME in perfusion MRI showed a significant decrease at week 1 and week 2 as compared with the baseline (p < 0.01). There was a more than 50% decrease in ME at week 1 in ON+ group; whereas there were no detectable ON lesions by conventional MRI at week 1, though 93% (14/15) rabbits could be detected at week 2 in ON+ group. In ON− group, ME showed a slight decrease at week 1 (less than 30%), and nearly recovered to normal at week 2 as compared with the baseline. Histological results showed a much larger average marrow fat area and more severe marrow blood sinusoids compression from surrounding crowded fat cells, and stronger positive TF expression in marrow endothelium and more thrombus formation in ON+ rabbits than ON− rabbits. This study demonstrated that functional perfusion MRI could predict development of steroid‐associated ON. Our experimental data suggested that perfusion MRI might be a sensitive noninvasive modality for monitoring steroid‐associated ON in patients.

Mesenchymal stem cell intracellular labeling using silica-coated superparamagnetic iron oxide nanoparticles with amine functional peripheries

Two types of superparamagnetic iron oxide nanoparticles designed for mesenchymal stem cell labeling and magnetic resonance imaging in vivo tracking were constructed in our laboratories. Both types of nanoparticles have an iron oxide core and a thin layer of silica coating, one of them has additional surface modification with amine. The overall particle size was around 8 nanometers. The in vitro study results demonstrated that, without the application of a transfection agent, both nanoparticles could be incorporated into bone marrow derived mesenchymal stem cells and located in lysosomes. Surface modification with amine significantly increased mesenchymal stem cell labeling efficiency, leading to an increased magnetic resonance imaging detection sensitivity of 2-5 folds.