Thimerosal, a competitive thioredoxin reductase 1 (TrxR1) inhibitor discovered via high-throughput screening.

Thioredoxin reductase 1 (TrxR1) is considered as an important anti-cancer drug target, inhibition of which can induce reactive oxygen species (ROS)-mediated apoptosis of human cancer cells. Here, we developed and optimized a high-throughput screening (HTS) assay based on enzyme kinetics for the discovery of TrxR1 inhibitors. By utilizing this assay, we performed a HTS for 2500 compounds from an in-house library against TrxR1. We found that a vaccine preservative, thimerosal, strongly inhibited TrxR1 in a competitive and reversible manner with an IC50 of 24.08 ± 0.86 nM. In addition, we determined that thiomersal has an inhibitory effect on the proliferation of A549 lung cancer cell line, with a GI50 of 6.81 ± 0.09 µM, slightly more potent than auranofin (GI50 = 11.85 ± 0.56 µM). Furthermore, we showed by flow cytometer that thimerosal effectively increased the content of ROS in A549 cells. Therefore, our work provided a high-throughput screening assay to quickly and effectively discover TrxR1 inhibitors, identifying thiomersal as a novel TrxR1 inhibitor and chemical probe.

The Th17/Treg cell balance: crosstalk among the immune system, bone and microbes in periodontitis.

Periodontopathic bacteria constantly stimulate the host, which causes an immune response, leading to host-induced periodontal tissue damage. The complex interaction and imbalance between Th17 and Treg cells may be critical in the pathogenesis of periodontitis. Furthermore, the RANKL/RANK/OPG system plays a significant role in periodontitis bone metabolism, and its relationship with the Th17/Treg cell imbalance may be a bridge between periodontal bone metabolism and the immune system. This article reviews the literature related to the Th17/Treg cell imbalance mediated by pathogenic periodontal microbes, and its mechanism involving RANKL/RANK/OPG in periodontitis bone metabolism, in an effort to provide new ideas for the study of the immunopathological mechanism of periodontitis.

Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

Design, synthesis of novel benzimidazole derivatives as ENL inhibitors suppressing leukemia cells viability via downregulating the expression of MYC.

Eleven-Nineteen-Leukemia Protein (ENL) containing YEATS domain, a potential drug target, has emerged as a reader of lysine acetylation. SGC-iMLLT bearing with benzimidazole scaffold was identified as an effective ENL inhibitor, but with weak activity against mixed-lineage leukemia (MLL)-rearranged cells proliferation. In this study, a series of compounds were designed and synthesized by structural optimization on SGC-iMLLT. All the compounds have been evaluated for their ENL inhibitory activities. The results showed that compounds 13, 23 and 28 are the most potential ones with the IC50 values of 14.5 ± 3.0 nM, 10.7 ± 5.3 nM, and 15.4 ± 2.2 nM, respectively, similar with that of SGC-iMLLT. They could interact with ENL protein and strengthen its thermal stability in vitro. Among them, compound 28 with methyl phenanthridinone moiety replacement of indazole in SGC-iMLLT, exhibited significantly inhibitory activities towards MV4-11 and MOLM-13 cell lines with IC50 values of 4.8 µM and 8.3 µM, respectively, exhibiting ∼7 folds and ∼9 folds more potent inhibition of cell growth than SGC-iMLLT. It could also increase the ENL thermal stability while SGC-iMLLT had no obvious effect on leukemia cells. Moreover, compound 28 could downregulate the expression of target gene MYC either alone or in combination with JQ-1 in cells, which was more effective than SGC-iMLLT. Besides, in vivo pharmacokinetic studies showed that the PK properties for compound 28 was much improved over that of SGC-iMLLT. These observations suggested compound 28 was a potential ligand for ENL-related MLL chemotherapy.

The role of Nrf2 in periodontal disease by regulating lipid peroxidation, inflammation and apoptosis.

Nuclear factor E2-related factor 2(Nrf2) is a transcription factor that mainly regulates oxidative stress in the body. It initiates the expression of several downstream antioxidants, anti-inflammatory proteins and detoxification enzymes through the Kelch-like ECH-associating protein 1 (Keap1) -nuclear factor E2-related factor 2(Nrf2) -antioxidant response element (ARE) signaling pathway. Its anti-apoptosis, anti-oxidative stress and anti-inflammatory effects have gradually become the focus of periodontal disease research in recent years. In this paper, the structure and function of Nrf2 pathway and its mechanism of action in the treatment of periodontitis in recent years were analyzed and summarized, so as to further clarify the relationship between Nrf2 pathway and oxidative stress in the occurrence and development of periodontitis, and to provide ideas for the development of new treatment drugs targeting Nrf2 pathway.

Salivary Microbiome Profile of Diabetes and Periodontitis in a Chinese Population.

Aim: There is a bidirectional association between diabetes and periodontitis. However, the effect of diabetes on the periodontitis salivary microbiota has not been elucidated. The aim of this study was to determine the effect of the presence of diabetes on the microbiota among Chinese patients with periodontitis. Materials and Methods: Unstimulated whole saliva samples were collected from the periodontitis with diabetes group (TC), chronic periodontitis group (CP), and periodontally healthy and systemically healthy group (H) by spitting method. Bacterial genomic DNA was PCR-amplified at the V4 variable region of 16S rRNA gene. The library was constructed according to the obtained sequence results, and biological analysis and statistical analysis were carried out. Functional prediction of three groups of microbial communities was performed by the PICRUSt algorithm. Results: There was no significant difference in bacterial diversity between the TC and CP groups. Compared with the H group, the TC group and CP group presented a higher diversity of salivary flora. Firmicutes, Streptococcus, Haemophilus, Veillonella, and Haemophilus parainfluenzae dominated the H group. Corynebacterium, Leptotrichia, Dialister, Comamonas, Capnocytophaga, Catonella, Filifactor, Campylobacter, Treponema, Campylobacter concisus, Prevotella oralis, and Porphyromonas gingivalis were significantly enriched in the TC and CP groups. Among them, Treponema and P. oralis were the most abundant in the TC group. The PICRUSt results showed that many pathways related to cell motility and functional metabolism of the salivary microbial flora changed in the TC group and the CP group. Conclusions: Diabetes was not the main factor causing the altered diversity of salivary microbiota in patients with periodontitis; however, the presence of diabetes altered the abundance of some microbiota in saliva.

Ferroptosis: A New Development Trend in Periodontitis.

Periodontitis is a chronic inflammatory disease associated with bacterial biofilm. It is characterized by loss of periodontal support tissue and has long been considered as a "silent disease". Because it is difficult to prevent and has a health impact that can not be ignored, researchers have been focusing on a mechanism-based treatment model. Ferroptosis is an iron-dependent regulatory form of cell death, that directly or indirectly affects glutathione peroxidase through different signaling pathways, resulting in a decrease in cell antioxidant capacity, accumulation of reactive oxygen species and lipid peroxidation, which cause oxidative cell death and tissue damage. Recently, some studies have proven that iron overload, oxidative stress, and lipid peroxidation exist in the process of periodontitis. Based on this, this article reviews the relationship between periodontitis and ferroptosis, in order to provide a theoretical reference for future research on the prevention and treatment of periodontal disease.

Sonodynamic effects of hematoporphyrin monomethyl ether on CNE-2 cells detected by atomic force microscopy.

Hematoporphyrin monomethyl ether (HMME) has been effectively used to treat solid tumors of some types. However, its application in nasopharyngeal carcinoma has not been studied yet. In this paper, the detailed sonodynamic effects of HMME-SDT (sonodynamic therapy) on CNE-2 cells including cell growth inhibition, apoptosis induction, and membrane toxicity were investigated. It was found that HMME alone had less cytotoxicity whereas HMME-SDT could suppress the cell proliferation in a dose-dependent manner as detected by MTT assay. The annexin V-based flow cytometric data indicated that upon SDT, different concentrations of HMME induce distinct types of cell death, apoptosis by low concentration (60 µg/ml) of HMME and necrosis by higher concentration (120 µg/ml). The immunofluorescence of cytoskeleton and nuclei morphology showed that upon HMME-SDT, the cells became rounding and the cytoskeletal network disappeared, and, the nuclei represented a total fragmented morphology of nuclear bodies. These alternations showed the apoptosis induction by HMME-SDT. Further AFM study showed that the cell membrane structure and cytoskeleton networks were destroyed, and, the Young's modulus, tip-cell-surface adhesion force decreased to 0.22 ± 0.11 Mpa, 35.4 ± 12.8 pN of cells with 120 µg/ml HMME-SDT from 0.48 ± 0.21 Mpa, 69.6 ± 22.3 pN of native cells, respectively. These membrane changes caused the collapse of mitochondrial transmembrane potential and disturbance of intracellular calcium homeostasis, which was consistent with the results detected by flow cytometry. Therefore, membrane toxicity and cytoskeleton disrupture induced by HMME-SDT maybe important factors to induce cell apoptosis, and, the disturbance of mitochondrial transmembrane potential and calcium channels might be the apoptosis mechanisms.

Detection of erythrocytes influenced by aging and type 2 diabetes using atomic force microscope.

The pathophysiological changes of erythrocytes are detected at the molecular scale, which is important to reveal the onset of diseases. Type 2 diabetes is an age-related metabolic disorder with high prevalence in elderly (or old) people. Up to now, there are no treatments to cure diabetes. Therefore, early detection and the ability to monitor the progression of type 2 diabetes are very important for developing effective therapies. Type 2 diabetes is associated with high blood glucose in the context of insulin resistance and relative insulin deficiency. These abnormalities may disturb the architecture and functions of erythrocytes at molecular scale. In this study, the aging- and diabetes-induced changes in morphological and biomechanical properties of erythrocytes are clearly characterized at nanometer scale using atomic force microscope (AFM). The structural information and mechanical properties of the cell surface membranes of erythrocytes are very important indicators for determining the healthy, diseased or aging status. So, AFM may potentially be developed into a powerful tool in diagnosing diseases.

AFM- and NSOM-based force spectroscopy and distribution analysis of CD69 molecules on human CD4+ T cell membrane.

Although CD69 is well known as an early T cell-activation marker, the possibility that CD69 are distributed as nano-structures on membrane for immune regulation during T cell activation has not been tested. In this study, nanoscale features of CD69 expression on activated T cells were determined using the atomic force microscopy (AFM) topographic and force-binding nanotechnology as well as near-field scanning optical microscopy (NSOM)-/fluorescence quantum dot (QD)-based nanosacle imaging. Unstimulated CD4(+) T cells showed neglectable numbers of membrane CD69 spots binding to the CD69 Ab-functinalized AFM tip, and no detectable QD-bound CD69 as examined by NSOM/QD-based imaging. In contrast, Phytohemagglutinin (PHA)-activated CD4(+) T cells expressed CD69, and displayed many force-binding spots binding to the CD69 Ab-functionalized AFM tip on about 45% of cell membrane, with mean binding-rupture forces 276 +/- 71 pN. Most CD69 molecules appeared to be expressed as 100-200 nm nanoclusters on the membrane of PHA-activated CD4(+) T cells. Meanwhile, NSOM/QD-based nanoscale imaging showed that CD69 were non-uniformly distributed as 80-200 nm nanoclusters on cell-membrane of PHA-activated CD4(+) T cells. This study represents the first demonstration of the nano-biology of CD69 expression during T cell activation.

Time-dependent surface adhesive force and morphology of RBC measured by AFM.

Atomic force microscopy (AFM) is a rapidly developing tool recently introduced into the evaluation of the age of bloodstains, potentially providing legal medical experts useful information for forensic investigation. In this study, the time-dependent, morphological changes of red blood cells (RBC) under three different conditions (including controlled, room-temperature condition, uncontrolled, outdoor-environmental condition, and controlled, low-temperature condition) were observed by AFM, as well as the cellular viscoelasticity via force-vs-distance curve measurements. Firstly, the data indicate that substrate types have different effects on cellular morphology of RBC. RBC presented the typical biconcave shape on mica, whereas either the biconcave shape or flattened shape was evident on glass. The mean volume of RBCs on mica was significantly larger than that of cells on glass. Surprisingly, the adhesive property of RBC membrane surfaces was substrate type-independent (the adhesive forces were statistically similar on glass and mica). With time lapse, the changes in cell volume and adhesive force of RBC under the controlled room-temperature condition were similar to those under the uncontrolled outdoor-environmental condition. Under the controlled low-temperature condition, however, the changes in cell volume occurred mainly due to the collapse of RBCs, and the curves of adhesive force showed the dramatic alternations in viscoelasticity of RBC. Taken together, the AFM detections on the time-dependent, substrate type-dependent, environment (temperature/humidity)-dependent changes in morphology and surface viscoelasticity of RBC imply a potential application of AFM in forensic medicine or investigations, e.g., estimating age of bloodstain or death time.

Effects of etchant on the nanostructure of dentin: an atomic force microscope study.

The objective of this study is to investigate effects of etchant on dentin surface as a function of etching time by atomic force microscopy (AFM). Twenty intact, freshly extracted noncarious human teeth were used to make 40 dentin discs and the discs were randomly divided into 4 groups. A commercial etchant was applied on these dentin discs. The main component of the etchant is 32% phosphoric acid and the etching time for the four dentin disc groups was 0, 20, 40, and 60 s. The AFM results show progressive changes of the surface morphology as the etching time increases. Significant difference of average roughness (Ra) exists in the dentin surface among all four groups (p<0.05). The statistic difference of diameters of dentinal tubule orifice (Dt) exists between the control group and all other groups (p<0.05), whereas the Dts for the 40-s group and 60-s group are not statistically different (p>0.05). Our results showed that acid treatment has a significant influence on dentin demineralization and the effective etching time of the dentin surface appears to be 60 s. We provide a new nanoscale insight into the dentin surface treatment and this can help us to select the optimal etching time in clinic.

The analysis of morphological distortion during AFM study of cells.

Atomic force microscopy (AFM) has been widely applied in cellular morphology study. However, morphological information including volume and roughness obtained by AFM are usually affected by different kinds of factors, which include the microscopic system itself, imaging mode, or external factors such as AFM probe or tip condition. In this study, based on red blood cell model, the dependence of cellular morphology, volume, and roughness on several parameters of the imaging was evaluated and, furthermore, a general rule and resolution for trustful analysis had been suggested. In addition, the potential effects that resulted from sample itself had also been analyzed based on adhesive force analysis. The results indicated that the scanning range and the imaging mode affect cellular volume and roughness, and the distorted images should be ascribed to blunt tip, contaminated tip, and the shape of tip. The analysis of morphological distortion during AFM investigation of cells provides a reference for researchers using AFM.

Correlation of serum adipocytokine levels with glycolipid metabolism and inflammatory factors in obese patients with periodontal disease.

OBJECTIVE: To analyze the correlation of serum levels of visfatin, leptin, resistin, and adiponectin (APN) with glycolipid metabolism and inflammatory factors in obese patients with periodontal disease. METHODS: 116 obese adults (OB), of whom 78 participants were diagnosed with different degrees of chronic periodontitis (CP), and 50 healthy adults were recruited into the study. Fasting peripheral venous blood was extracted to determine serum levels of adipocytokines (e.g., visfatin, leptin, resistin, and APN), glucolipid metabolism (e.g., fasting blood glucose (FBG), fasting insulin (FINS), C-peptide (C-P), cortisol (Cor), homeostasis model of assessment for insulin resistance index (HOMA-IR), glycosylated hemoglobin (HbA1c), triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), and non-high-density lipoprotein cholesterol (non-HDL)), and micro-inflammation-related indexes like C-reactive protein (CRP), interleukin (IL)-1ß, IL-6, IL-10, and tumor necrosis factor (TNF)-α. Correlation between levels of adipocytokines and levels of glucolipid metabolism and inflammatory factors was further analyzed. RESULTS: Assays for plasma levels of adipocytokines showed that both the OB group and the OB with CP group had significantly higher serum levels of visfatin, leptin, and resistin than the normal control group and significantly lower serum levels of ANP than the normal control group (P<0.05). Detection of serum glucolipid metabolism levels showed that FBG, FINS, C-P, Cor, HOMA-IR, TG, TC, LDL-C, HDL-C, Non-HDL-C of OB group, and OB with CP patients were significantly higher than those of normal patients (P<0.05). Assay for plasma levels of inflammatory factors showed that both the OB group and the OB with CP group had significantly higher serum levels of CRP, IL-1ß, IL-6, and TNF-α than the normal control group and significantly lower serum levels of IL-10 than the normal control group. Spearman's correlation analysis revealed that serum levels of visfatin, leptin, resistin, and APN were significantly correlated with concentrations of FBG, FINS, C-P, Cor, TG, TC, LDL-C, HDL-C, Non-HDL-C, CRP, IL-1ß, IL-6, IL-10, and TNF-α. CONCLUSIONS: There were high expression levels of inflammatory factors and glucolipid metabolism disorder in obese patients with periodontal disease and excessively expressed adipocytokines may be important factors of persistent and worsened obesity and of periodontitis.

Research on Mechanisms and Controlling Methods of Macro Defects in TC4 Alloy Fabricated by Wire Additive Manufacturing.

Wire feeding additive manufacturing (WFAM) has broad application prospects because of its advantages of low cost and high efficiency. However, with the mode of lateral wire feeding, including wire and laser additive manufacturing, gas tungsten arc additive manufacturing etc., it is easy to generate macro defects on the surface of the components because of the anisotropy of melted wire, which limits the promotion and application of WFAM. In this work, gas tungsten arc additive manufacturing with lateral wire feeding is proposed to investigate the mechanisms of macro defects. The results illustrate that the defect forms mainly include side spatters, collapse, poor flatness, and unmelted wire. It was found that the heat input, layer thickness, tool path, and wire curvature can have an impact on the macro defects. Side spatters are the most serious defects, mainly because the droplets cannot be transferred to the center of the molten pool in the lateral wire feeding mode. This research indicates that the macro defects can be controlled by optimizing the process parameters. Finally, block parts without macro defects were fabricated, which is meaningful for the further application of WFAM.

Effect of Molten Pool Size on Microstructure and Tensile Properties of Wire Arc Additive Manufacturing of Ti-6Al-4V Alloy.

Wire arc additive manufacturing (WAAM) technique is a cost-competitive and efficient technology to produce large structure components in industry domains. Mechanical properties are mainly dominated by the microstructure of the components, which is deeply affected by the molten pool size. In this work, to investigate the effect of the molten pool size on microstructure and mechanical properties of the components, a series of Ti-6Al-4V alloy blocks with different width of molten pool (WMP) ranging from 7 mm to 22 mm were deposited by adjusting the wire feed speed (WFS) from 100 cm/min to 500 cm/min. It is interesting to find that the macrostructure changes from columnar grains to equiaxial grains, and then returns to large columnar grains with the increase of WMP, which is mainly caused by the different cooling rates and thermal gradients. Nonetheless, the tensile properties of the components have a tendency to decline with the increase of WMP.

Graded Nano Glass-Zirconia Material for Dental Applications-Part II Biocompatibility Evaluation.

A graded glass/graded zirconia (G/Z) system was previously synthesized via the infiltration of a low modulus nanosized glass into a zirconia surface. The bond strength of G/Z to veneering porcelains was demonstrated to be 3-fold higher than in zirconia-based systems. Nevertheless, biocompatibility testing prior to the possible clinical application of G/Z systems is essential. Herein, such biocompatibility testing was performed with L-929 fibroblasts seeded onto G/Z and yttria-stabilized tetragonal zirconia polycrystals (Y-TZP) for 2-72 h. Assessments included an oral mucous membrane irritation test in conjunction with analyses of cell viability, cell morphology, cell cycle, cell apoptosis, oxidative stress responses, inflammatory cytokine expression, and cellular toxicity. Cell viability tests showed no significant decrease in G/Z- and Y-TZP-treated cells over 72 h. Fluorescence and SEM images demonstrated that cell spreading on Y-TZP and G/Z was similar; cells were flattened and well-spread. Oxidative stress data for G/Z- and Y-TZP-treated cells showed no significant difference in ROS production. Cellular toxicity results for G/Z did not elicit significant differences in LDH release compared with Y-TZP over 72 h. G/Z and Y-TZP had no significant differences in IL-1α, IL-8, PGE2, and TNF-α expression and elicited significantly increased IL-8 expression compared with that of the untreated control group. Cells that were cultured with G/Z showed no significant changes in cell cycle distribution compared with Y-TZP or the untreated control group. Cells that were cultured with Y-TZP and G/Z showed no apoptosis compared to untreated controls at 24 and 48 h. According to the oral mucous membrane irritation test, scores for the macroscopic and microscopic observations for both G/Z and Y-TZP sides were 0, demonstrating no consequent irritation. Therefore, the excellent biocompatibility of G/Z indicates that it has potential for future clinical applications.

Performance of High Layer Thickness in Selective Laser Melting of Ti6Al4V.

To increase building rate and save cost, the selective laser melting (SLM) of Ti6Al4V with a high layer thickness (200 µm) and low cost coarse powders (53 µm-106 µm) at a laser power of 400 W is investigated in this preliminary study. A relatively large laser beam with a diameter of 200 µm is utilized to produce a stable melt pool at high layer thickness, and the appropriate scanning track, which has a smooth surface with a shallow contact angle, can be obtained at the scanning speeds from 40 mm/s to 80 mm/s. By adjusting the hatch spacings, the density of multi-layer samples can be up to 99.99%, which is much higher than that achieved in previous studies about high layer thickness selective laser melting. Meanwhile, the building rate can be up to 7.2 mm³/s, which is about 2 times-9 times that of the commercial equipment. Besides, two kinds of defects are observed: the large un-melted defects and the small spherical micropores. The formation of the un-melted defects is mainly attributed to the inappropriate overlap rates and the unstable scanning tracks, which can be eliminated by adjusting the processing parameters. Nevertheless, the micropores cannot be completely eliminated. It is worth noting that the high layer thickness plays a key role on surface roughness rather than tensile properties during the SLM process. Although a sample with a relatively coarse surface is generated, the average values of yield strength, ultimate tensile strength, and elongation are 1050 MPa, 1140 MPa, and 7.03%, respectively, which are not obviously different than those with the thin layer thickness used in previous research; this is due to the similar metallurgical bonding and microstructure.

Synergistic effect of RhBMP-2 and bFGF on ectopic osteogenesis in mice.

OBJECTIVE: To investigate the synergistic effect and mechanism of the combined application of recombinant human bone morphogenetic protein-2 (rhBMP-2) and basic fibroblast growth factor (bFGF). METHODS: 24 KM male mice were randomly divided into 6 groups with 4 mice in each group, namely, Group A (control group), Group B (only treated with collagen), Group C (treated with 2 ng bFGF+collagen), Group D (treated with 4 µ g rhBMP-2+collagen), Group E (treated with 4 µ g rhBMP-2+2 ng bFGF+collagen) and Group F (treated with 4 µ g rhBMP-2+4 ng bFGF+collagen). The composites were implanted into the intermuscular septum of hind legs mice; whereas in control group, intermuscular septum of mice was separated and no implantation was performed. General observation, detection of concentration of calcium content, micro computed tomography (Micro-CT), three-dimensional reconstruction scan, measurement of bone mineral density (BMD), bone volume fraction (BVF) and trabecular thickness (Tb.Th), as well as histological observation with HE staining and ALP and CD34 immumohistochemical staining were performed. RESULTS: Ectopic osteogenesis was found in Groups D, E and F mice. The difference in concentration of calcium contents was statistically significant between Groups D and E (P<0.05), but insignificant between Groups E and F (P>0.05). Micro-CT and three-dimensional reconstruction revealed continuous newborn bone substance in external surface of ectopic bone formation, and the center of bone formation did not show obvious substantial filling by bone substance. The differences in BMD, BVF and Tb.Th were statistically significant between Groups D and E or F (P<0.01 or <0.05). HE staining showed that in Groups D, E and F, newborn bone substance was mainly located at the edge of ectopic bone formation, and the bone formation in Groups E and F was better than that in Group D. ALP and CD34 immumohistochemical staining revealed the positive expression mainly at the edge of ectopic bone formation, and area of positive expression in Groups E and F was larger than that in Groups D. CONCLUSIONS: rhBMP-2 possesses the capacity to induce ectopic osteogenesis independently, but bFGF does not have this ability; the combined application of rhBMP-2 and bFGF can enhance the synergetic effect on inducing ectopic osteogenesis.