OBJECTIVES: To investigate the effect of recombinant human fibroblast growth factor 21 (rhFGF21) on the proliferation and mineralization of cementoblasts and its mechanism. METHODS: Hematoxylin eosin, immunohistochemical staining, and immunofluorescence were used to detect the expression and distribution of fibroblast growth factor 21 (FGF21) in rat periodontal tissues and cementoblasts (OCCM-30), separately. Cell Counting Kit-8 was used to detect the proliferation of OCCM-30 under treatment with rhFGF21. Alkaline phosphatase staining and Alizarin Red staining were used to detect the mineralization state of OCCM-30 after 3 and 7 days of mineralization induction. The transcription and protein expression of the osteogenic-related genes Runx2 and Osterix were detected by real-time quantitative polymerase chain reaction (PCR) and Western blot analysis. The expression levels of genes of transforming growth factor β (TGFβ)/bone morphogenetic protein (BMP) signaling pathway in OCCM-30 were detected through PCR array analysis. RESULTS: FGF21 was expressed in rat periodontal tissues and OCCM-30. Although rhFGF21 had no significant effect on the proliferation of OCCM-30, treatment with 50 ng/mL rhFGF21 could promote the mineralization of OCCM-30 cells after 7 days of mineralization induction. The transcriptional levels of Runx2 and Osterix increased significantly at 3 days of mineralization induction and decreased at 5 days of mineralization induction. Western blot analysis showed that the protein expression levels of Runx2 and Osterix increased during mineralization induction. rhFGF21 up-regulated Bmpr1b protein expression in cells. CONCLUSIONS rhFGF21 can promote the mineralization ability of OCCM-30. This effect is related to the activation of the TGFβ/BMP signaling pathway.
Humans ; Rats ; Animals ; Dental Cementum ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Cell Differentiation ; Bone Morphogenetic Proteins/metabolism* ; Transforming Growth Factor beta/pharmacology*

This study compared the effects on bone metabolism and morphology of pathological obesity induced by excessive fat intake in a non-hibernator (mice) versus healthy obesity due to pre-hibernation fattening in a hibernator (ground squirrels). Kunming mice were fed a high-fat diet to provide a model of pathological obesity (OB group). Daurian ground squirrels fattened naturally in their pre-hibernation season (PRE group) were used as a healthy obesity model. Micro-computed tomography (micro-CT) and three-point bending tests were used to determine the microstructure and mechanical properties of bone. Western blots were used to analyze protein expression levels related to bone metabolism (Runt-related transcription factor 2 (RunX2), osteocalcin (OCN), alkaline phosphatase (ALP), osteoprotegerin (OPG), receptor activator of nuclear factor-‍κB ligand (RANKL), cathepsin K, matrix metallopeptidase 9 (MMP9), patched protein homolog 1 (Ptch1), phosphorylated β‍-‍catenin (P‍-‍β‍-‍catenin), and glycogen synthase kinase-3β (GSK-3β)). Compared with controls, there was no obvious bone loss in the OB mice, and the stiffness of the femur was increased significantly. Compared with summer active squirrels, bone formation was enhanced but the mechanical properties did not change in the PRE group squirrels. In OB mice, western blots showed significantly increased expression levels of all proteins except RunX2, OPG, and Ptch1. PRE ground squirrels showed significantly increased expression of most proteins except OCN and Ptch1, which decreased significantly, and P‍-‍β‍-‍catenin and OPG, which did not change. In conclusion, for non-hibernating mice, moderate obesity had a certain protective effect on bones, demonstrating two-way regulation, increasing both bone loss and bone formation. For pre-hibernating ground squirrels, the healthy obesity acquired before hibernation had a positive effect on the microstructure of bones, and also enhanced the expression levels of proteins related to bone formation, bone resorption, and Wnt signaling.
Mice ; Animals ; Hibernation ; Core Binding Factor Alpha 1 Subunit/metabolism* ; Glycogen Synthase Kinase 3 beta/metabolism* ; Diet, High-Fat ; X-Ray Microtomography ; Sciuridae/metabolism* ; Obesity

OBJECTIVE: To explore effect of tobramycin (TOB) on healing of femoral fractures in rats. METHODS: Totally 32 male sprague-dawley (SD) rats were selected and randomly divided into sham group (group A), fracture group (group B), fracture with TOB group (group C) and fracture + TOB + IWR-1 group (group D), 8 rats in each group. Close femoral fracture model in rats were established in group B, C and D, group A was sham operation without otherwise process. Group D was intraperitoneal injected 100 μl (8 μM) of Wnt pathway inhibitor IWR-1-endo (IWR-1) before molding at 1 day. At 1 day after molding, 100 μl (100 μM) of TOB was intraperitoneally injected into group C and D at once a day for 7 days. At 7 weeks after modling, fracture healing of group B, C and D were observed by X-ray, Western blotting was appilied to detect alkaline phosphatase(ALP) and Runt related transcription factor 2 (RUNX2) and β-catenin of Wnt passway. RESULTS: X-ray results showed fracture line disappeared, callus formation and fracture healing well in group C compared with begning of molding; while a little fracture line, callus formation and fracture malunion in group B and d could be seen. Western blotting results showed ALP, RUNX2 and expression of β-catenin in group B, C and D were higher than that of group A ( CONCLUSION Tobramycin could promote osteoblast differentiation and fracture healing by stimulating Wnt / β-catenin signaling pathway, up regulating expression of ALP and RUNX2.
Alkaline Phosphatase ; Animals ; Cell Differentiation ; Core Binding Factor Alpha 1 Subunit/genetics* ; Femoral Fractures ; Fracture Healing ; Male ; Osteogenesis ; Rats ; Tobramycin ; Wnt Signaling Pathway ; beta Catenin/metabolism*

BACKGROUND: Cytogenetic abnormalities are one of the most reliable prognostic factors in acute leukemia. Combination of conventional chromosome analysis (CCA) and FISH provides higher sensitivity in detecting these genetic abnormalities, and it is effective to apply several FISH probes as a profile test. The objective of this study was to investigate the utility of FISH profile analyses in the initial diagnosis of acute leukemia. METHODS: Two hundred and forty one de novo acute leukemia patients diagnosed from January, 2002 to November, 2007 were included. For acute lymphoblastic leukemia profile test, FISH probes for BCR/ABL, TEL/AML1, MLL gene rearrangement and CDKN2A deletion were used. For acute myeloid leukemia profile test, probes for AML1/ETO, MLL and CBFbeta gene rearrangement were used. The results of CCA and FISH profile tests were collected, and the positive rates were compared. RESULTS: ALL FISH profile tests revealed additional genetic aberrations not detected by chromosome analysis in 48.6% (67/138) of cases, including those with normal karyotypes or no mitotic cells (37%, 51/138). Among these 51 cases, TEL/AML1 abnormalities were detected in 44.3%, followed by the abnormal CDKN2A signal (24.6%) and hyperdiploidy (18.0%). AML FISH profile tests revealed additional genetic abnormalities in 7.8% (8/103) of cases. CONCLUSIONS: FISH analysis as a profile test detected additional genetic aberrations in a significant proportion of acute leukemia, and was effective especially in detecting cryptic translocations, submicroscopic deletions and complex karyotypes. Our study supports the need to incorporate FISH profile test at initial work up in acute leukemia.
Adolescent ; Adult ; Child ; Child, Preschool ; *Chromosome Aberrations ; Core Binding Factor Alpha 2 Subunit/genetics ; Core Binding Factor beta Subunit/genetics ; Cyclin-Dependent Kinase Inhibitor p16/genetics ; Female ; Humans ; In Situ Hybridization, Fluorescence/*methods ; Infant ; Infant, Newborn ; Karyotyping ; Leukemia, Myeloid, Acute/*diagnosis/genetics ; Male ; Middle Aged ; Myeloid-Lymphoid Leukemia Protein/genetics ; Precursor Cell Lymphoblastic Leukemia-Lymphoma/*diagnosis/genetics ; Proto-Oncogene Proteins c-bcr/genetics

OBJECTIVETo investigate the probabilities of core-biding factor a1 (Cbfa1) expression by human skin fibroblasts induced in vitro.

METHODSThe fibroblasts were isolated, purified from human skin, and were grown in incubation in the media of TNF-alpha, BMP-2, and combined TNF-alpha and BMP-2 at certain concentrations, respectively. The changes in biological features of these fibroblasts correlated with osteogenesis were detected by immunohistochemistry and RT-PCR assay.

RESULTSTNF-alpha could switch phenotype of collagen in fibroblasts from Type I and III to Type I and induce fibroblasts to express Ras and BMP type I receptor (BMPR-IA). TNF-alpha in combination with BMP-2 could induce fibroblasts to express Cbfa1 and osteocalcin mRNA.

CONCLUSIONHuman skin fibroblast could be induced into pro-osteoblast expressing Cbfa1, an osteoblast-specific transcription factor and a regulation of osteoblast differentiation, and combined use of TNF-alpha and BMP-2 was one of the regulating factors.

Bone Morphogenetic Protein 2 ; Bone Morphogenetic Protein Receptors, Type I ; Bone Morphogenetic Proteins ; pharmacology ; Cells, Cultured ; Collagen ; biosynthesis ; Core Binding Factor Alpha 1 Subunit ; Core Binding Factors ; Fibroblasts ; metabolism ; Humans ; Neoplasm Proteins ; Osteocalcin ; biosynthesis ; Protein-Serine-Threonine Kinases ; biosynthesis ; RNA, Messenger ; analysis ; Receptors, Growth Factor ; biosynthesis ; Skin ; cytology ; Transcription Factors ; biosynthesis ; genetics ; Transforming Growth Factor beta ; Tumor Necrosis Factor-alpha ; pharmacology

This study was purposed to establish new method for detecting CBFB-MYH11 fusion gene in acute myeloid leukemia (AML) and to evaluate its value in clinical use. All fusion types of reported CBFB-MYH11 fusion gene were defined by search of references and databank, then the primers and probes were designed on this basis, and 3 positive plasmids and negative cell line as control were established. GUSB gene was also amplified as an internal reference. The primer/probe sets were tested with 3 positive plasmids and HL-60 cDNA using quantitative real-time PCR (qPCR) assays, which were then combined as a multiplex qPCR for simultaneous detection of CBFB-MYH11 and GUSB. After optimization, the multiplex qPCR assay demonstrated both high sensitivity (10 copies for all the 3 plasmids) and high specificity. Finally, the multiplex qPCR assay was clinically evaluated with 58 AML patients, and 4 CBFB-MYH11-positive cases (6.9%) were detected, involving A type (3 cases) and J type (1 case). By comparison, the multiplex qPCR assay showed results concordant with sequencing results, and detected one case that was missed by cytogenetic analysis. It is concluded that a novel qPCR method for screening of CBFB-MYH11 fusion gene in AML is established. This method is fast, comprehensive, sensitive, specific, reliable, and should consider to be a robust tool for identification and management of AML patients with CBFB-MYH11 fusion gene.
Case-Control Studies ; Core Binding Factor beta Subunit ; analysis ; genetics ; HL-60 Cells ; Humans ; Leukemia, Myeloid, Acute ; diagnosis ; genetics ; Myosin Heavy Chains ; analysis ; genetics ; Oncogene Proteins, Fusion ; analysis ; genetics ; Real-Time Polymerase Chain Reaction

OBJECTIVE: To compare a new thermosensitive recombinant human amelogenin (rhAm) carrier and traditional propylene glycol alginate (PGA) carrier for their characteristics, antibacterial activity, and biocompatibility with human periodontal membrane fibroblasts. METHODS: PGA-rhAm was prepared by mixing 3.3% PGA and rhAm, and CS-βGP-rhAm was prepared by mixing 2% chitosan (CS) with rhAm and then with 60% β-sodium glycerophosphate solution (βGP) as the crosslinking agent. The biophysical properties of the prepared carriers were characterized, and their antibacterial activity was assessed by observing Staphylococcus aureus growth. The biocompatibility of the carriers was evaluated in human periodontal membrane fibroblasts (hPDLFs) using CCK8 assay and scratch test, and mRNA and protein expressions of osteogenic genes of the cells incubated with the carriers were detected using RT-qPCR and Western blotting; osteogenic differentiation of the cells was detected using alkaline phosphatase staining. RESULTS: PGA-rhAm had a viscosity value of 3.262±0.055 Pa.s. CS-βGP-rhAm had a solidification capacity of 6 min at 37 ℃ with a pH value close to that of the oral cavity and a swelling rate of about 90%. CS-β GP-rhAm maintained sustained release of rhAm for over 2 weeks with a self-degradation time over 3 weeks. CS-βGPrhAm more effectively inhibited the growth of S. aureus than rhAm-loaded PGA. While PGA did not obviously affect the proliferation of hPDLFs, both CS-βGP and CS-βGP-rhAm significantly promoted the cell proliferation(P < 0.001). Scratch test showed that after rhAm loading, both CS-βGP and PGA promoted cell migration (P < 0.01). CS-βGP-rhAm significantly enhanced the mRNA expressions of RUNX2 and OCN mRNA level and the protein expressions of Ki67, RUNX2, collagen I, and β-catenin (P < 0.05); PGA-rhAm only enhanced RUNX2 (P < 0.05) and OCN (P < 0.01) mRNA expressions without significant effects on the protein expressions. Alkaline phosphatase staining results showed that CS-βGP, but not PGA, promoted osteogenic differentiation of hPDLFs. CONCLUSION CS-βGP carrier is capable of sustained release of rhAm, inhibiting the growth of S. aureus, and improving the biological activity of hPDLFs without affecting the bioactivity of rhAm after drug loading.
Alginates ; Alkaline Phosphatase ; Amelogenin ; Anti-Bacterial Agents/pharmacology* ; Cell Differentiation ; Cells, Cultured ; Chitosan/pharmacology* ; Collagen ; Core Binding Factor Alpha 1 Subunit ; Delayed-Action Preparations ; Glycerophosphates ; Humans ; Ki-67 Antigen ; Osteogenesis ; Periodontal Ligament ; RNA, Messenger ; Staphylococcus aureus ; beta Catenin

AIMUnderstanding the response of mesenchymal stem cells (MSCs) to mechanical strain and their consequent gene expression patterns will broaden our knowledge of the mechanobiology of distraction osteogenesis.

METHODOLOGYIn this study, a single period of cyclic mechanical stretch (0.5 Hz, 2,000 microepsilon) was performed on rat bone marrow MSCs. Cellular proliferation and alkaline phosphatase (ALP) activity was examined. The mRNA expression of six bone-related genes (Ets-1, bFGF, IGF-II, TGF-beta, Cbfa1 and ALP) was detected using real-time quantitative RT-PCR.

RESULTSThe results showed that mechanical strain can promote MSCs proliferation, increase ALP activity, and up-regulate the expression of these genes. A significant increase in Ets-1 expression was detected immediately after mechanical stimulation, but Cbfa1 expression became elevated later. The temporal expression pattern of ALP coincided perfectly with Cbfa1.

CONCLUSIONThe results of this study suggest that mechanical strain may act as a stimulator to induce differentiation of MSCs into osteoblasts, and that these bone-related genes may play different roles in the response of MSCs to mechanical stimulation.

Alkaline Phosphatase ; analysis ; Animals ; Antigens, Surface ; analysis ; Biomechanical Phenomena ; Bone Marrow Cells ; physiology ; Cell Culture Techniques ; Cell Differentiation ; physiology ; Cell Proliferation ; Core Binding Factor Alpha 1 Subunit ; analysis ; Fibroblast Growth Factor 2 ; analysis ; Insulin-Like Growth Factor II ; analysis ; Mesenchymal Stromal Cells ; physiology ; Osteoblasts ; physiology ; Osteogenesis, Distraction ; Pluripotent Stem Cells ; physiology ; Proto-Oncogene Protein c-ets-1 ; analysis ; Rats ; Stress, Mechanical ; Transforming Growth Factor beta ; analysis ; Up-Regulation ; physiology