OBJECTIVEPolyethylene glycol/bone morphogenetic protein-2 (PEG/BMP-2) nanoparticles were transfected into Rabbit bone mesenchymal stem cells (rBMSCs) and the expression of BMP-2 was detected.

METHODSDissociated rBMSCs were primarily cultured in vitro and BMP-2 gene was transfected into rBMSCs by PEG/BMP-2 nanoparticals and lipofectamine, respectively. The efficiency of transfection was detected by flow cytometry and the expression of BMP-2 was detected by Western Blot and real time RT-PCR.

RESULTSPEG/BMP-2 nanoparticals were successfully synthesized and transfected into rBMSCs. Compared with the lipofectamine transfection group, PEG/BMP-2 transfection group had higher efficiency and higher BMP-2 expression.

CONCLUSIONPEG/BMP-2 nanoparticals transfected rBMSCs highly expressed BMP-2,which provided novel strategies for the treatment of bone defect.

Animals ; Bone Diseases ; genetics ; therapy ; Bone Morphogenetic Protein 2 ; chemistry ; genetics ; metabolism ; Bone and Bones ; cytology ; Gene Expression Regulation ; Humans ; Mesenchymal Stromal Cells ; cytology ; metabolism ; Nanoparticles ; chemistry ; Polyethylene Glycols ; chemistry ; RNA, Messenger ; genetics ; metabolism ; Rabbits ; Transfection ; methods

The temporal expression of estrogen receptor (ER)-alpha and ER-beta mRNA was examined in male Japanese quails. Femurs of quails receiving 17beta-estradiol underwent RTPCR and histochemical analysis 1 to 15 days after treatment. Untreated quails were used as controls (day 0). Between days 0 and 5, cells lining the bone endosteal surface differentiated into osteoblasts, which in turn formed medullary bone. Expression of ER-alpha was already observed on day 0 and increased slightly during bone formation whereas ER-beta was hardly detected throughout this process. After osteoclasts appeared on the medullary bone surface, this type of bone disappeared from the bone marrow cavity (days 7~15). ER-alpha expression simultaneously decreased slightly and ER-beta levels remained very low. These results suggest that estrogen activity mediated by ER-alpha not only affects medullary bone formation but also bone resorption.
Animals ; Bone Resorption/genetics ; Bone and Bones/chemistry/cytology/*metabolism ; Cells, Cultured ; Coturnix/*metabolism ; Estradiol/*pharmacology ; Estrogen Receptor alpha/genetics/*metabolism ; Estrogen Receptor beta/genetics/*metabolism ; Gene Expression Regulation ; Male ; Osteoblasts/chemistry/cytology/*metabolism ; Osteogenesis/genetics ; RNA, Messenger/metabolism ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo observe the function of wnt/β-catenin signal pathway on the process that epimedium-derived flavonoids (EFs) regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, and to provide an experimental evidence for the mechanism of EFs on treating postmenopausal osteoporosis.

METHODSBone marrow stromal cells from ovariectomized rats were separated and cultivated in the condition of osteoinductive medium or liquid medium for 15 days. Low- (1 μg/mL), medium- (10 μg/mL) and high- (100 μg/mL) dose EFs were administrated correspondingly. Alkaline phosphatase (ALP) staining, ALP activity determination, oil red O staining and realtime polymerese chain reaction (RT-PCR) were used to determine the effect of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats. Moreover, in order to explore the mechanism of EFs on osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats, Dickkopf-related protein 1 (DKK1) was used in the medium group. Enzymelinked immunosorbent assay (ELISA) and RT-PCR were used to determine mRNA levels of β-catenin, low density lipoprotein receptor-related protein 5 (LRP5) and T cell factor (TCF) protein, known as wnt/β-catenin signal pathway related factors.

RESULTSEFs increased mRNA expression levels of ALP and early osteoblast differentiation factors, such as runt-related transcription factor 2 (Runx2), osteocalcin and collagen I, and decreased mRNA expression levels of fat generation factors, such as peroxisome proliferator activated receptor gamma 2 (PPARγ-2) and CCAAT enhancer-binding protein-α (C/EBPα) in a dose-dependent manner. While osteoblast differentiation factors were down-regulated, fat generation factors were up-regulated when DKK1 was applied. Also EFs up-regulated mRNA expression levels of β-catenin, LRP5 and TCF protein which could be blocked by DKK1.

CONCLUSIONEFs regulate the balance between osteogenic differentiation and adipogenic differentiation in bone marrow stromal cells of ovariectomized rats by activating wnt/β-catenin signal pathway, which may be an important molecular mechanism of EFs on treating postmenopausal osteoporosis.

Adipose Tissue ; cytology ; drug effects ; metabolism ; Animals ; Base Sequence ; Bone and Bones ; cytology ; drug effects ; metabolism ; Cell Differentiation ; drug effects ; DNA Primers ; Enzyme-Linked Immunosorbent Assay ; Epimedium ; chemistry ; Female ; Flavonoids ; pharmacology ; Flow Cytometry ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Rats ; Real-Time Polymerase Chain Reaction ; Signal Transduction ; Wnt Proteins ; metabolism ; beta Catenin ; metabolism