mi R -15a/15b Cluster Modulates Survival of Mesenchymal Stem Cells to Improve Its Therapeutic Efficacy of Myocardial Infarction.

Background The poor viability of transplanted mesenchymal stem cells (MSCs) hampers their therapeutic efficacy for ischemic heart disease. Micro RNA s are involved in regulation of MSC survival and function. The present study was designed to investigate the molecular effects of mi R -15a/15b on MSC survival, focusing on the role of vascular endothelial growth factor receptor 2. Methods and Results We first harvested donor luc(Luciferase)- MSC s (5×105) isolated from the luciferase transgenic mice with FVB background. Luc- MSC s were transfected with miR-15a/15b mimics or inhibitors and cultured under oxygen glucose deprivation condition for 12 hours to mimics the harsh microenvironment in infarcted heart; they were subjected to MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide?Thiazolyl Blue Tetrazolium Bromide) assay, bioluminescence imaging, quantitative reverse transcription-polymerase chain reaction, transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling assay, and flow cytometry. Furthermore, the levels of vascular endothelial growth factor receptor 2, protein kinase B, p(Phosphorylate)-protein kinase B, Bcl-2, Bax, and caspase-3 proteins were available by Western blotting assay. In vivo, acute myocardial infarction was induced in 24 mice by coronary ligation, with subsequent receipt of Luc- MSC s, Luc- MSC s+miR-15a/15b inhibitors, or PBS treatment. The therapeutic procedure and treatment effects were tracked and assessed using bioluminescence imaging and echocardiographic measurement. Next, ex vivo imaging and immunohistochemistry were conducted to verify the distribution of MSC s. We demonstrated that miR-15a/15b targeted vascular endothelial growth factor receptor 2 to modulate MSC survival, possibly via phosphatidylinositol 3-kinase/protein kinase B signaling pathway, which was proved by bioluminescence imaging, immunohistochemistry analysis, and echocardiographic measurement. Conclusions Luc- MSC s could be followed dynamically in vitro and in vivo by bioluminescence imaging, and the role of mi R -15a/b could be inferred from the loss of signals from luc- MSC s. This finding may have practical clinical implications in mi R -15a/15b-modified MSC transplantation in treating myocardial infarction.

Baicalin alleviates ischemia-induced memory impairment by inhibiting the phosphorylation of CaMKII in hippocampus.

Baicalin has a significant neuroprotective effect in stroke. However, the mechanism remains unclear. This study was to reveal the mechanisms by which baicalin protected hippocampal neurons and improved learning and memory impairment after global cerebral ischemia/reperfusion in gerbil. In the present study, the Morris water maze test showed that baicalin significantly improved learning and memory impairment after global cerebral ischemia/reperfusion in gerbils. Laser scanning confocal fluorescence microscope examination showed that baicalin suppressed OGD-induced augmentation of intracellular calcium concentration. Western blotting analysis indicated that baicalin suppressed ischemia-caused elevated phosphorylation level of CaMKII in vivo, in hippocampal neurons in culture, and in SH-SY5Y cells in culture. Western blotting, TUNEL and RNA interference technology were applied to detect effects of baicalin on neuronal apoptosis. We found that baicalin, a CaMKII inhibitor and knocking down the CaMKII prevented OGD-induced apoptosis of hippocampal or SH-SY5Y cells in culture. Therefore, these results suggested that baicalin improves learning and memory impairment induced by global cerebral ischemia/reperfusion in gerbils via attenuating the phosphorylation level of CaMKII and further preventing hippocampal neuronal apoptosis.

In vitro and in vivo enhancement of osteogenic capacity in a synthetic BMP-2 derived peptide-coated mineralized collagen composite.

Enhancement of osteogenic capacity was achieved in a mineralized collagen composite, nano-hydroxyapatite/collagen (nHAC), by loading with synthetic peptides derived from BMP-2 residues 32-48 (P17-BMP-2). Rabbit marrow stromal cells (MSCs) were used in vitro to study cell biocompatibility, attachment and differentiation on the mineralized collagen composite by a cell counting kit, scanning electron microscopy (SEM) and real-time reversed transcriptase-polymerase chain reaction analysis (RT-PCR). Optimal peptide dosage (1.0 µg/mL) was obtained by RT-PCR analysis in vitro. In addition, the relative expression level of OPN and OCN was significantly upregulated on P17-BMP-2/nHAC compared with nHAC. In vitro results of P17-BMP-2 release kinetics demonstrated that nHAC released P17-BMP-2 in a controlled and sustained manner. In the rabbit mandibular box-shaped bone defect model, osteogenic capacity of three groups (nHAC, P17-BMP-2/nHAC, rhBMP-2/nHAC) was evaluated. Compared to the nHAC group, bone repair responses in both P17-BMP-2/nHAC and rhBMP-2/nHAC group implants were significantly improved based on histological analysis. The osteogenic response of the P17-BMP-2/nHAC group was similar to that of the rhBMP-2/nHAC group.

Injectable biocomposites for bone healing in rabbit femoral condyle defects.

A novel biomimetic bone scaffold was successfully prepared in this study, which was composed of calcium sulfate hemihydrate (CSH), collagen and nano-hydroxyapatite (nHAC). CSH/nHAC was prepared and observed with scanning electron microscope and rhBMP-2 was introduced into CSH/nHAC. The released protein content from the scaffold was detected using high performance liquid chromatography at predetermined time interval. In vivo bone formation capacity was investigated by means of implanting the scaffolds with rhBMP-2 or without rhBMP-2 respectively into a critical size defect model in the femoral condyle of rabbit. The releasing character of rhBMP-2 was that an initial burst release (37.5%) was observed in the first day, followed by a sustained release and reached 100% at the end of day 20. The CSH/nHAC showed a gradual decrease in degradation with the content of nHAC increase. The results of X-rays, Micro CT and histological observation indicated that more new bone was formed in rhBMP-2 group. The results implied that this new injectable bone scaffold should be very promising for bone repair and has a great potential in bone tissue engineering.