Kerong Dai

The clinical use of enriched bone marrow stem cells combined with porous beta-tricalcium phosphate in posterior spinal fusion

Cytotherapy for bone regeneration has not been widely used clinically. A new method based on enriched bone-marrow-derived mesenchymal stem cells (MSCs) combined with porous beta-tricalcium phosphate (beta-TCP) was used for posterior spinal fusion in 41 patients. The aim of the present study was to assess the clinical feasibility of peri-operative bone marrow stem cell enrichment and their combination with tricalcium phosphate. About 252 ml marrow per patient was harvested from bilateral iliac crest, the enriched MSCs were produced by a cell processor peri-operatively, then combined with porous beta-TCP granules by a negative pressure and a short-time incubation in the meantime of conventional operation, which were finally implanted back into the patient. About 45 ml enriched MSC suspension was collected, and 78+/-16% of MSCs were recovered. By enrichment technique, the number of colony-forming units which expressed alkaline phosphatase (CFUs-ALP+, to estimate the prevalence of MSCs) was increased 4.3 times; the increasing folds of bone marrow nucleated cells (NCs) and MSCs had a positive correlation. The natural log (ln) of MSC number declined with age, and also, the MSC number of younger subjects (< or =40 years) was more than that of older ones (>40 years), but none for NCs. The number of NCs and MSCs was not different significantly between men and women. However, the patients with thoracolumbar fracture (TLF) had significantly more MSCs than those with degenerative disc disease (DDD), but not for NCs. On the other hand, enriched MSCs could adhere to the wall of porous beta-TCP within 2h combination, and proliferate well during culture in vitro. After 34.5 months, 95.1% cases had good spinal fusion results. None of the samples before grafting was positive in bacterial culture. Only four patients had a little exudation or moderate swelling in their wounds, and recovered with conservative treatment.

Uptake mechanisms of non-viral gene delivery.

Non-viral gene delivery is currently a hot subject for its relative safety and simplicity of use; however, it is still far from being ideal enough to be clinically used for its comparatively lower efficiency than viral gene delivery. To improve the efficiency of non-viral gene delivery needs a comprehensive understanding of the uptake mechanisms. Macromolecules are internalized into cells by a variety of mechanisms, and their intracellular fates are usually relevant with the uptake pathways. The uptake pathways of non-viral gene complexes are usually determined by not only the gene/carrier interaction but also by the interaction between complexes and target cells. The best-characterized uptake pathway is the so-called clathrin-mediated endocytic pathway. However, there are numerous updates of knowledge about endocytic pathways and even non-endocytic pathways in recent years with the development of novel technologies for tracking and inhibiting. In this review, we will try to sort out our current understanding of the uptake mechanisms of non-viral gene delivery. In addition, factors for pathway selection are summarized in the third section. Finally, the useful inhibitors or tools for the study of these pathways will also be concluded in the last section.

Silicate bioceramics induce angiogenesis during bone regeneration.

The capacity to induce rapid vascular ingrowth during new bone formation is an important feature of biomaterials that are to be used for bone regeneration. Akermanite, a Ca-, Mg- and Si-containing bioceramic, has been demonstrated to be osteoinductive and to promote bone repair. This study further demonstrates the ability of akermanite to promote angiogenesis and investigates the mechanism of this behavior. The akermanite ion extract predominantly caused Si-ion-stimulated proliferation of human aortic endothelial cells. The Si ion in the extract was the most important component for the effect and the most effective concentration was found to be 0.6-2 μg ml(-1). In this range of Si ion concentration, the stimulating effect of the ceramic ion extract was demonstrated by the morphology of cells at the primary, interim and late stages during in vitro angiogenesis using ECMatrix™. The akermanite ion extract up-regulated the expression of genes encoding the receptors of proangiogenic cytokines and also increased the expression level of genes encoding the proangiogenic downstream cytokines, such as nitric oxide synthase and nitric oxide synthesis. Akermanite implanted in rabbit femoral condyle model promoted neovascularization after 8 and 16 weeks of implantation, which further confirmed its stimulation effect on angiogenesis in vivo. These results indicate that akermanite ceramic, an appropriate Si ion concentration source, could induce angiogenesis through increasing gene expression of proangiogenic cytokine receptors and up-regulated downstream signaling. To our knowledge, akermanite ceramic is the first Si-containing ceramic demonstrated to be capable of inducing angiogenesis during bone regeneration.

Proliferation and Osteoblastic Differentiation of Human Bone Marrow Stromal Cells on Hydroxyapatite/Bacterial Cellulose Nanocomposite Scaffolds

In this study, we prepared hydroxyapatite/bacterial cellulose (HAp/BC) nanocomposite scaffolds utilizing the biomimetic technique, and investigated the proliferation and osteoblastic differentiation of stromal cells derived from human bone marrow (hBMSC) on them. Scanning electron microscopy proved that cells could adhere and spread on scaffolds. The hBMSC seeded on the nanocomposites exhibited better adhesion and activity than those seeded upon the pure BC. After 6 days of culture on scaffolds, the cells proliferated faster on the nanocomposites than on the pure BC, as assessed by Alamar Blue assay. Real-time reverse transcription PCR results showed that the alkaline phosphatase (ALP) activity of hBMSC and the expression of osteopontin, osteocalcin, bone sialoprotein, and ALP mRNA were all higher for up to 7 days for hBMSC cultured on the nanocomposites than for those cultured upon the pure BC with and without the presence of osteogenic supplements (L-ascorbic acid, glycerophosphate, and dexamethasone, p<0.05). These results suggest that the attachment, proliferation, and differentiation in cultured hBMSC can be modulated by the HAp/BC nanocomposite scaffold properties. In summary, we have developed a scaffold that displays in vitro biocompatibility, which may have potential use for bone tissue engineering.

Repairing of goat Tibial Bone Defects with BMP-2 Gene–Modified Tissue-Engineered Bone

Bone defects larger than a critical size are major challenges in orthopedic medicine. We combined tissue-engineered bone and gene therapy to provide osteoprogenitor cells, osteoinductive factors, and osteoconductive carrier for ideal bone regeneration in critical-sized bone defects. Goat diaphyseal bone defects were repaired with tissue and genetically engineered bone implants, composed of biphasic calcined bone (BCB) and autologous bone marrow derived mesenchymal stem cells (BMSC) transduced with human bone morphogenetic protein-2 (hBMP-2). Twenty six goats with tibial bone defects were divided into groups receiving implants by using a combination of BCB and BMSCs with or without the hBMP-2 gene. In eight goats that were treated with BCB that contained hBMP-2 transduced BMSC, five had complete healing and three showed partial healing. Goats in other experimental groups had only slight or no healing. Furthermore, the area and biochemical strength of the callus in the bone defects were significantly better in animals treated with genetically engineered implants. We concluded that the combination of genetic and tissue engineering provides an innovative way for treating critical-sized bone defects.

Stimulation of osteogenic differentiation and inhibition of adipogenic differentiation in bone marrow stromal cells by alendronate via ERK and JNK activation

To elucidate the mechanism of the effect of bisphosphonates on bone metabolism, we investigated the effect of alendronate, a widely used bisphosphonate, on osteogenic and adipogenic differentiation in bone marrow stromal cells (BMSCs) derived from ovariectomized SD rats. Alendronate treatment not only increased the mRNA level of bone morphogenetic protein-2, runt-related transcription factor 2, osteopontin, bone sialoprotein, and alkaline phosphatase activity after osteogenic induction, but also decreased the mRNA level of peroxisome proliferator activated receptor gamma 2 and total droplet number indicated by Oil Red O staining after adipogenic induction. The effect of alendronate treatment was dose-dependent, and the difference of the osteogenic or the adipogenic potential between the treated group and the non-treated group was statistically significant (p<0.001). The MAPK-specific inhibitors, PD98059 and SP600125, but not the p38-specific inhibitor, blocked the alendronate-induced regulation of BMSC differentiation. Analysis of BMSCs induced in the presence of alendronate revealed an immediate increase in ERK and JNK phosphorylation. Taken together, these data suggest that alendronate acts on BMSCs to stimulate osteogenic differentiation and inhibit adipogenic differentiation in a dose-dependent manner; this effect is mediated via activating ERK and JNK.

Magnesium and the Risk of Cardiovascular Events: A Meta-Analysis of Prospective Cohort Studies

Background Prospective studies that have examined the association between dietary magnesium intake and serum magnesium concentrations and the risk of cardiovascular disease (CVD) events have reported conflicting findings. We undertook a meta-analysis to evaluate the association between dietary magnesium intake and serum magnesium concentrations and the risk of total CVD events. Methodology/Principal Findings We performed systematic searches on MEDLINE, EMBASE, and OVID up to February 1, 2012 without limits. Categorical, linear, and nonlinear, dose-response, heterogeneity, publication bias, subgroup, and meta-regression analysis were performed. The analysis included 532,979 participants from 19 studies (11 studies on dietary magnesium intake, 6 studies on serum magnesium concentrations, and 2 studies on both) with 19,926 CVD events. The pooled relative risks of total CVD events for the highest vs. lowest category of dietary magnesium intake and serum magnesium concentrations were 0.85 (95% confidence interval 0.78 to 0.92) and 0.77 (0.66 to 0.87), respectively. In linear dose-response analysis, only serum magnesium concentrations ranging from 1.44 to 1.8 mEq/L were significantly associated with total CVD events risk (0.91, 0.85 to 0.97) per 0.1 mEq/L (Pnonlinearity = 0.465). However, significant inverse associations emerged in nonlinear models for dietary magnesium intake (Pnonlinearity = 0.024). The greatest risk reduction occurred when intake increased from 150 to 400 mg/d. There was no evidence of publication bias. Conclusions/Significance There is a statistically significant nonlinear inverse association between dietary magnesium intake and total CVD events risk. Serum magnesium concentrations are linearly and inversely associated with the risk of total CVD events.

A meta-analysis of 18FDG-PET–CT, 18FDG-PET, MRI and bone scintigraphy for diagnosis of bone metastases in patients with lung cancer

BACKGROUND AND PURPOSE Lung cancer is the most common cause of cancer related death among both men and women worldwide. The skeleton is the most common site of cancer metastasis. Early detection is crucial for prognosis. To evaluate and compare the capability for bone metastasis assessment of [(18)F] fluoro-2-d-glucose positron emission tomography with computed tomography ((18)FDG-PET-CT), [(18)F] fluoro-2-d-glucose positron emission tomography ((18)FDG-PET), magnetic resonance imaging (MRI) and bone scintigraphy (BS) in lung cancer patients, a meta-analysis is preformed. METHODS We searched MEDLINE, OVID, EMBASE and the Cochrane Library for studies evaluating diagnosis validity of (18)FDG-PET-CT, (18)FDG-PET, MRI and BS between January 1990 and August 2010. Meta-analysis methods were used to pool sensitivity, specificity, diagnostic odd ratios (DORs) and to construct a summary receiver-operating characteristic curve (SROC). RESULTS A total of 17 articles (9 (18)FDG-PET-CT studies, 9 (18)FDG-PET studies, 6 MRI studies and 16 BS studies) that included 2940 patients who fulfilled all of the inclusion criteria were considered for inclusion in the analysis. The pooled sensitivity for the detection of bone metastasis in lung cancer using (18)FDG-PET-CT, (18)FDG-PET, MRI and BS were 0.92 (95% CI, 0.88-0.95), 0.87 (95% CI, 0.81-0.92), 0.77 (95% CI, 0.65-0.87) and 0.86 (95% CI, 0.82-0.89), respectively. The pooled specificity for the detection of bone metastasis from lung cancer using (18)FDG-PET-CT, (18)FDG-PET, MRI and BS were 0.98 (95% CI, 0.97-0.98), 0.94 (95% CI, 0.92-0.96), 0.92 (95% CI, 0.88-0.95), 0.88 (95% CI, 0.86-0.89), respectively. The pooled DORs estimates for (18)FDG-PET-CT 449.17 were significantly higher than for (18)FDG-PET (118.25, P<0.001), MRI (38.27, P<0.001) and BS (63.37, P<0.001). The pooled sensitivity of BS was not correlated with the prevalence of bone metastasis. CONCLUSION The results showed that both (18)FDG-PET-CT and (18)FDG-PET were better imaging methods for diagnosing bone metastasis from lung cancer than MRI and BS. (18)FDG-PET-CT has higher diagnostic value (sensitivity, specificity and DORs) for diagnosing bone metastasis from lung cancer than any other imaging methods.

Effect of berberine on Staphylococcus epidermidis biofilm formation

Staphylococcus epidermidis is one of the main causes of medical device-related infections owing to its adhesion and biofilm-forming abilities on biomaterial surfaces. Berberine is an isoquinoline-type alkaloid isolated from Coptidis rhizoma (huang lian in Chinese) and other herbs with many activities against various disorders. Although the inhibitory effects of berberine on planktonic bacteria have been investigated in a few studies, the capacity of berberine to inhibit biofilm formation has not been reported to date. In this study, we observed that berberine is bacteriostatic for S. epidermidis and that sub-minimal inhibitory concentrations of berberine blocked the formation of S.epidermidis biofilm. Using viability assays and berberine uptake testing, berberine at a concentration of 15-30mug/mL was shown to inhibit bacterial metabolism. Data from this study also indicated that modest concentrations of berberine (30-45mug/mL) were sufficient to exhibit an antibacterial effect and to inhibit biofilm formation significantly, as shown by the tissue culture plate (TCP) method, confocal laser scanning microscopy and scanning electron microscopy for both S. epidermidis ATCC 35984 and a clinical isolate strain SE243. Although the mechanisms of bacterial killing and inhibition of biofilm formation are not fully understood, data from this investigation indicated a potential application for berberine as an adjuvant therapeutic agent for the prevention of biofilm-related infections.

The promotion of cartilage defect repair using adenovirus mediated Sox9 gene transfer of rabbit bone marrow mesenchymal stem cells

Although Sox9 is essential for chondrogenic differentiation and matrix production, its application in cartilage tissue engineering has been rarely reported. In this study, the chondrogenic effect of Sox9 on bone marrow mesenchymal stem cells (BMSCs) in vitro and its application in articular cartilage repair in vivo were evaluated. Rabbit BMSCs were transduced with adenoviral vector containing Sox9. Toluidine blue, safranin O staining and real-time PCR were performed to check chondrogenic differentiation. The results showed that Sox9 could induce chondrogenesis of BMSCs both in monolayer and on PGA scaffold effectively. The rabbit model with full-thickness cartilage defects was established and then repaired by PGA scaffold and rabbit BMSCs with or without Sox9 transduction. HE, safranin O staining and immunohistochemistry were used to assess the repair of defects by the complex. Better repair, including more newly-formed cartilage tissue and hyaline cartilage-specific extracellular matrix and greater expression of several chondrogenesis marker genes were observed in PGA scaffold and BMSCs with Sox9 transduction, compared to that without transduction. Our findings defined the important role of Sox9 in the repair of cartilage defects in vivo and provided evidence that Sox9 had the potential and advantage in the application of tissue engineering.