BACKGROUND15-Deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2), one of the major metabolites from prostaglandin D2 in arachidonic acid metabolic pathway, has potential anti-inflammatory properties. The objective of this study was to explore the effects of 15d-PGJ2-loaded poly(D,L-lactide-co-glycolide) nanocapsules (15d-PGJ2-NC) on inflammatory responses and bone regeneration in local bone defect.

METHODSThe study was conducted on 96 Wistar rats from June 2014 to March 2016. Saline, unloaded nanoparticles, free 15d-PGJ2or 15d-PGJ2-NC, were delivered through a collagen vehicle inside surgically created transcortical defects in rat femurs. Interleukin-6 (IL-6), interleukin-1 beta (IL-1β), and tumor necrosis factor-alpha (TNF-α) levels in the surrounding soft tissue were analyzed by Western blot and in the defect by quantitative real-time polymerase chain reaction over 14 days. Simultaneously, bone morphogenetic protein-6 (BMP-6) and platelet-derived growth factor-B (PDGF-B) messenger RNA (mRNA) in the defect were examined. New bone formation and EphrinB2 and osteoprotegerin (OPG) protein expression in the cortical defect were observed by Masson's Trichrome staining and immunohistochemistry over 28 days. Data were analyzed by one-way analysis of variance. Least-significant difference and Dunnett's T3 methods were used with a bilateral P< 0.05.

RESULTSApplication of l5d-PGJ2-NC (100 μg/ml) in the local bone defect significantly decreased IL-6, IL-1β, and TNF-α mRNA and protein, compared with saline-treated controls (P < 0.05). l5d-PGJ2-NC upregulated BMP-6 and PDGF-B mRNA (P < 0.05). New bone formation was observed in the cortical defect in l5d-PGJ2-NC-treated animals from 7th day onward (P < 0.001). Expression of EphrinB2 and OPG presented early on day 3 and persisted through day 28 in 15d-PGJ2-NC group (P < 0.05).

CONCLUSIONStable l5d-PGJ2-NC complexes were prepared that could attenuate IL-6, IL-1β, and TNF-α expression, while increasing new bone formation and growth factors related to bone regeneration.

Animals ; Bone Morphogenetic Protein 6 ; metabolism ; Bone Regeneration ; drug effects ; Inflammation ; drug therapy ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Male ; Platelet-Derived Growth Factor ; metabolism ; Prostaglandin D2 ; analogs & derivatives ; therapeutic use ; Rats ; Rats, Wistar ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the expression of follistatin-like protein 1 (FSTL-1) in bone metastasis of prostate cancer (BMPC), the correlation of serum FSTL-1 with the chronic inflammatory factor interleukin-6 (IL-6) and bone morphogenetic protein 6 (BMP6) , and the clinical application value of serum FSTL-1 in BMPC.

METHODSUsing ELISA, we measured the expression levels of serum FSTL-1, IL-6, and BMP6 in 35 patients with BMPC and another 30 with benign prostatic hyperplasia (BPH) and performed correlation analysis on the data obtained.

RESULTSCompared with the BPH controls, the BMPC patients showed a significantly decreased expression of serum FSTL-1 ([34.45 ± 12.35] μg/L vs [20.23 ± 8.69] μg/L, P < 0.01) and increased levels of IL-6 ([11.21 ± 8.62] μg/L vs [23.56 ± 20.12] μg/L, P < 0.05) and BMP6 ([293.50 ± 39.72] μg/L vs [428.30 ± 178.40] μg/L, P < 0.05). There was a significant negative correlation between the level of serum FSTL-1 and those of IL-6 and BMP6 in the BMPC patients, with correlation coefficients of -0.971 and -0.972, respectively (P < 0.05).

CONCLUSIONThe expression of serum FSTL-1 decreases in patients with bone metastasis of prostate cancer, and it is correlated with the levels of inflammatory factor and cell transformation factor. This finding offers a novel biological marker for the development and progression of prostate cancer as well as a new biological target factor for its intervention.

Aged ; Biomarkers, Tumor ; blood ; Bone Morphogenetic Protein 6 ; blood ; Bone Neoplasms ; blood ; secondary ; Disease Progression ; Follistatin-Related Proteins ; blood ; Humans ; Interleukin-6 ; blood ; Male ; Prostatic Hyperplasia ; blood ; Prostatic Neoplasms ; blood ; pathology

OBJECTIVETo construct a recombinant adenovirus co-expressing bone morphogenic protein (BMP) 9 and BMP6 and observe its effect on the osteogenesis in C3H10 cells.

METHODThe full-length sequences of BMP9 and BMP6 were amplified from AdEasy vector by PCR and cloned into the shuttle plasmid pASG2 vector to construct the co-expression shuttle plasmid pASG2-BMP9, 6 followed by homologous recombination with plasmid pAdeasy-1 in BJ5183. After confirmation by restriction endonuclease digestion, the recombinant vector was transfected into HEK293 cells, and high-titer recombinant adenovirus (Ad-BMP9, 6) was collected after amplification. Ad-BMP9, 6 was then transduced into C3H10 cells in vitro, and the mRNA expression of BMP9 and BMP6 was detected by RT-PCR. The osteogenic capability of the transfected cells was observed by alkaline phosphatase staining and calcium-alizarin red staining.

RESULTSAdBMP9,6 was constructed successfully and effectively infected in C3H10 cells, in which high expressions of BMP6 and BMP9 were detected. C3H10 cells infected with Ad-BMP9,6 showed stronger alkaline phosphatase and calcium-alizarin red staining than the cells transfected by either BMP9 or BMP6 alone.

CONCLUSIONThe recombinant adenovirus co-expressing BMP9 and BMP6 we constructed shows a more potent effect than the adenoviruses expressing either BMP9 or BMP6 alone in inducing the osteogenic differentiation of C3H10 cells into osteoblasts.

Adenoviridae ; genetics ; Bone Morphogenetic Protein 6 ; genetics ; Genetic Vectors ; Growth Differentiation Factors ; genetics ; HEK293 Cells ; Humans ; Osteoblasts ; cytology ; Osteogenesis ; Plasmids ; Recombinant Fusion Proteins ; genetics ; Transfection

The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 µg·L⁻¹ TGF-β3 or 100 µg∙L⁻¹ BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.
Adult Stem Cells ; physiology ; Aggrecans ; analysis ; Bone Morphogenetic Protein 6 ; pharmacology ; Cell Culture Techniques ; Cell Differentiation ; drug effects ; Cell Separation ; Chondrogenesis ; drug effects ; physiology ; Collagen Type II ; analysis ; Flow Cytometry ; Glycosaminoglycans ; analysis ; Humans ; Immunohistochemistry ; Mesenchymal Stromal Cells ; drug effects ; physiology ; Molar, Third ; cytology ; Periodontal Ligament ; cytology ; drug effects ; Reverse Transcriptase Polymerase Chain Reaction ; SOX9 Transcription Factor ; analysis ; Stress, Mechanical ; Tooth, Impacted ; pathology ; Transforming Growth Factor beta3 ; pharmacology

Dermal cells from neonatal mice can initiate the formation of hair follicles (HFs) when combined with adult mouse epidermal cells and transplanted subcutaneously into athymic mice. In the present study, the effects of dermal cells on HF formation were tested in terms of total cell number and the time course of cell harvest. Results demonstrated that the number of dermal cells is critical to the formation of HF. Furthermore, hair forming ability is rapidly decreasing as the neonatal mice age. To examine potential differences in gene expression, cDNA array was performed. Results demonstrate that numerous molecules which are directly involved in receptor and signaling correlated with decreased hair inductivity in early time points after delivery. It is reported that bone morphogenic protein (BMP)-6 and Wnt3a treatment increased hair inductivity of dermal papilla cells. But in our study, no changes were observed in the expression levels of BMP-6 and Wnt3a. However, several Wnt related genes demonstrate increased or decreased expression levels. Thus, our results suggest that co-ordinated regulation of these molecules will be important in hair neogenesis within our model system.
Adult ; Animals ; Bone Morphogenetic Protein 6 ; Cell Count ; Gene Expression ; Hair ; Hair Follicle ; Humans ; Infant, Newborn ; Mice ; Mice, Nude ; Oligonucleotide Array Sequence Analysis ; Transplants

OBJECTIVE: To investigate the effect of human bone morphogenetic protein (hBMPs) 2/3/6 and 12 on osteosarcoma cell UMR106. METHODS: Adenovirus-BMP2/3/6 and 12 (AdBMP2/3/6 and12) were used to treat the cell line. Their proliferation, apoptosis, and transmigration were detected by Trypan blue exclusion test, TdT-mediated biotinylated-dUTP nick end labeling (TUNEL), acridine orange-ethidium bromide (AO/EB) double fluorescent dye staining, and transwell-room test, respectively. The alkaline phosphatase (ALP) activity was detected to reflect the differentiation of tumors. RESULTS: Compared with the control groups, the cell survival rate of the experimental groups treated with AdBMP2/3/6 and 12 showed a significant time-dependent decrease (P<0.01). The apoptosis indexes were increased significantly (P<0.01) and the results from TUNEL and AO/EB method were consistent. The cell numbers of transmembrane significantly decreased at 24,48, and 72 h (P<0.01). AdBMP2/3/6 and 12 treatment enhanced the activity of ALP activity from day 3 and this effect might still be observed up to day 9 of the treatment (P<0.01). CONCLUSION hBMPs2/3/6 and 12 can inhibit the proliferation and transmigration, and induce their apoptosis and differentiation in osteosarcoma cell line UMR106.
Adenoviridae ; genetics ; metabolism ; Apoptosis ; drug effects ; Bone Morphogenetic Protein 2 ; pharmacology ; Bone Morphogenetic Protein 3 ; pharmacology ; Bone Morphogenetic Protein 6 ; pharmacology ; Bone Morphogenetic Proteins ; pharmacology ; Bone Neoplasms ; pathology ; Cell Line, Tumor ; Cell Transformation, Neoplastic ; drug effects ; Growth Differentiation Factors ; pharmacology ; Humans ; Osteosarcoma ; pathology ; Recombinant Proteins ; pharmacology

BACKGROUNDBone morphogenetic protein (BMP) is a member of the superfamily of transforming growth factor-beta. Recent studies show that it is an indispensable factor in hematopoiesis. To better characterize the effect of recombinant human BMP (rhBMP)-2 in hematopoiesis, we set out to determine whether rhBMP-2 could promote the proliferation of mesenchymal stem cells (MSCs) and increase the levels of hematopoietic cytokines in MSCs.

METHODS2, 3-bis (2-methoxy-4-nitro-5-sulfophenyl)-5-((phenylamino) carbonyl)-2H-tetrazolium hydroxide (XTT), real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) were used to detect the effects of rhBMP-2 on the proliferation and hematopoietic cytokine levels of MSCs. In addition, MSCs marked with Hoechst33342 were transplanted into BALB/c mice by the intravenous route or intra-bone marrow transplantation, and cluster numbers were counted.

RESULTSThe XTT test revealed that rhBMP-2 significantly induced proliferation of MSCs in doses ranging from 10 ng/ml to 0.1 mg/ml in a dose-dependent manner. The experiments in vivo showed that there were more clusters of donor cells in bone marrow, spleen, liver and lung of the BMP group than those in the control group after both intra-bone marrow transplantation (P < 0.001, P < 0.001, P < 0.001, and P = 0.001, respectively) and intravenous transplantation (P < 0.001, P < 0.001, and P < 0.001 respectively). The results of real-time PCR and ELISA revealed that rhBMP-2 significantly increased mRNA expressions and protein levels of IL-6, IL-7, IL-11, G-CSF, M-CSF and SCF.

CONCLUSIONSThe treatment with rhBMP-2 promotes the proliferation of MSCs in vivo and in vitro and increases the levels of hematopoietic cytokines in MSCs, which may contribute to the improvement of hematopoietic function.

Animals ; Bone Morphogenetic Protein 2 ; Bone Morphogenetic Proteins ; pharmacology ; Cell Proliferation ; drug effects ; Cells, Cultured ; Dose-Response Relationship, Drug ; Enzyme-Linked Immunosorbent Assay ; Granulocyte Colony-Stimulating Factor ; genetics ; Humans ; Interleukin-11 ; genetics ; Interleukin-6 ; genetics ; Interleukin-7 ; genetics ; Macrophage Colony-Stimulating Factor ; genetics ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Mice ; Mice, Inbred BALB C ; Polymerase Chain Reaction ; Recombinant Proteins ; pharmacology ; Stem Cell Factor ; genetics ; Transforming Growth Factor beta ; pharmacology

The objective of this research is to investigate the influence of rhBMP-2 on the renal tissue of rat with renal ischemia reperfusion injury. In this program the ischemia reperfusion rat model was established and Wistar rats were divided into six groups: sham operation group (S group), renal ischemia reperfusion injury group (R group), rhBMP-2 treatment group (B1, B2, B3 and B4 group). In the rhBMP treatment groups, rhBMP-2 was intravenously administered with different doses before reperfusion. The contents of TNF-alpha, IL-6, IL-8, MDA and SOD in kidney tissue were observed. At the same time, renal function (blood creatine (Scr) and urea nitrogen (BUN)) were measured. As a result, compared with renal ischemia reperfusion group, administration of rhBMP-2 significantly reduced the content of IL-6 and IL-8 (P < 0.05) and ameliorated renal dysfunction cellular damages (P < 0.05). Higher dose of rhBMP-2 may reduce the content of TNF-alpha (P < 0.05) in kidney tissue. rhBMP-2 also increased activity of SOD and reduced the level of MDA, BUN and Scr. So, we can draw a conclusion that rhBMP-2 treatment attenuates renal ischemia reperfusion injury through inhibition of pro-inflammatory cytokines production and anti-oxidation activity.
Adrenal Cortex ; pathology ; ultrastructure ; Animals ; Blood Urea Nitrogen ; Bone Morphogenetic Protein 2 ; pharmacology ; Creatine ; blood ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Kidney ; blood supply ; metabolism ; Male ; Malondialdehyde ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Recombinant Proteins ; pharmacology ; Reperfusion Injury ; metabolism ; pathology ; Superoxide Dismutase ; metabolism ; Transforming Growth Factor beta ; pharmacology ; Tumor Necrosis Factor-alpha ; metabolism

BMP6 is a potent protein for future treatment strategies of bone regeneration as it is a very important regulator of bone homeostasis. Active BMP6 is a dimer containing multidisulfide bonds and is a highly hydrophobic protein prone to aggregation. To obtain soluble and active BMP6 in Escherichia coli, we compared the effects of four N-terminal fusion tags (TRX, GST, MBP and CBD) and N-terminal His6-tag. The expression and solubility were tested under the different conditions (expression hosts, temperatures and inductor concentrations). A series of experiments leads to the finding that the placement of MBP before the BMP6 is best in availing the soluble expression of the protein. Our study alsodemonstrates that in E. coli BL21trxB(DE3) cytoplasm, which is a thioredoxin reductase mutant strain, soluble homodimeric BMP6 can be formed. The overexpressed MBP-BMP6 fusion protein is purified by chromatography, and shown to be functionally active.
Bone Morphogenetic Protein 6 ; biosynthesis ; genetics ; Carrier Proteins ; genetics ; Escherichia coli ; genetics ; metabolism ; Genetic Vectors ; Humans ; Maltose-Binding Proteins ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; isolation & purification ; metabolism ; Solubility ; Transformation, Bacterial

OBJECTIVE: To compare protein levels of pro-inflammatory factors and bone formation mediators in the fibrous cap and shoulder region of non-calcified and calcified carotid endarterectomy (CEA) plaques. METHODS: Twenty-two CEA plaques were classified as non-calcified and calcified groups (n=11 each) in accordance with the American Heart Association (AHA) consensus in 1995. To make frozen sections and H&E staining using plaque, the mean percent of carotid stenosis and calcification area was determined by quantitative histomorphometry. The protein levels of pro-inflammatory interleukin-8 (IL-8), monocyte chematactic protein-1 (MCP-1), bone formation mediators bone morphogenetic protein-6 (BMP-6), and osteocalcin in the fibrous cap and shoulder region of plaques were determined by western blot and were quantified using ImageJ software. RESULTS: MCP-1 and IL-8 protein were 1.3 (P>0.05) and 1.5 (P<0.05) folds greater in the non-calcified plaques than those in the calcified plaques. BMP-6 and osteocalcin protein were 1.3 (P>0.05) and 2.1 (P<0.01) folds greater in the calcified plaques compared with those of the non-calcified plaques. CONCLUSION Inflammation is more likely to occur in non-calcified carotid plaques, and calcification in the plaques may be associated with bone formation, which indicates that decreased inflammation may be the beginning of calcification in carotid atherosclerotic plaques.
Atherosclerosis ; complications ; metabolism ; pathology ; Bone Morphogenetic Protein 6 ; metabolism ; Calcinosis ; metabolism ; pathology ; Carotid Stenosis ; etiology ; metabolism ; pathology ; Chemokine CCL2 ; metabolism ; Endarterectomy, Carotid ; Humans ; Inflammation ; metabolism ; pathology ; Interleukin-8 ; metabolism