Osimertinib Covalently Binds to CD34 and Eliminates Myeloid Leukemia Stem/Progenitor Cells.

Osimertinib is a third-generation covalent EGFR inhibitor that is used in treating non-small cell lung cancer. First-generation EGFR inhibitors were found to elicit pro-differentiation effect on acute myeloid leukemia (AML) cells in preclinical studies, but clinical trials yielded mostly negative results. Here, we report that osimertinib selectively induced apoptosis of CD34+ leukemia stem/progenitor cells but not CD34- cells in EGFR-negative AML and chronic myeloid leukemia (CML). Covalent binding of osimertinib to CD34 at cysteines 199 and 177 and suppression of Src family kinases (SFK) and downstream STAT3 activation contributed to osimertinib-induced cell death. SFK and STAT3 inhibition induced synthetic lethality with osimertinib in primary CD34+ cells. CD34 expression was elevated in AML cells compared with their normal counterparts. Genomic, transcriptomic, and proteomic profiling identified mutation and gene expression signatures of patients with AML with high CD34 expression, and univariate and multivariate analyses indicated the adverse prognostic significance of high expression of CD34. Osimertinib treatment induced responses in AML patient-derived xenograft models that correlated with CD34 expression while sparing normal CD34+ cells. Clinical responses were observed in two patients with CD34high AML who were treated with osimertinib on a compassionate-use basis. These findings reveal the therapeutic potential of osimertinib for treating CD34high AML and CML and describe an EGFR-independent mechanism of osimertinib-induced cell death in myeloid leukemia. SIGNIFICANCE: Osimertinib binds CD34 and selectively kills CD34+ leukemia cells to induce remission in preclinical models and patients with AML with a high percentage of CD34+ blasts, providing therapeutic options for myeloid leukemia patients.

C1Q labels a highly aggressive macrophage-like leukemia population indicating extramedullary infiltration and relapse.

Extramedullary infiltration (EMI) is a concomitant manifestation that may indicate poor outcome of acute myeloid leukemia (AML). The underlying mechanism remains poorly understood and therapeutic options are limited. Here, we employed single-cell RNA sequencing on bone marrow (BM) and EMI samples from a patient with AML presenting pervasive leukemia cutis. A complement C1Q+ macrophage-like leukemia subset, which was enriched within cutis and existed in BM before EMI manifestations, was identified and further verified in multiple patients with AML. Genomic and transcriptional profiling disclosed mutation and gene expression signatures of patients with EMI that expressed high levels of C1Q. RNA sequencing and quantitative proteomic analysis revealed expression dynamics of C1Q from primary to relapse. Univariate and multivariate analysis demonstrated adverse prognosis significance of C1Q expression. Mechanistically, C1Q expression, which was modulated by transcription factor MAF BZIP transcription factor B, endowed leukemia cells with tissue infiltration ability, which could establish prominent cutaneous or gastrointestinal EMI nodules in patient-derived xenograft and cell line-derived xenograft models. Fibroblasts attracted migration of the C1Q+ leukemia cells through C1Q-globular C1Q receptor recognition and subsequent stimulation of transforming growth factor ß1. This cell-to-cell communication also contributed to survival of C1Q+ leukemia cells under chemotherapy stress. Thus, C1Q served as a marker for AML with adverse prognosis, orchestrating cancer infiltration pathways through communicating with fibroblasts and represents a compelling therapeutic target for EMI.

Effects of rhBMP-2 on Bone Formation Capacity of Rat Dental Stem/Progenitor Cells from Dental Follicle and Alveolar Bone Marrow.

Dental stem/progenitor cells are a promising cell sources for alveolar bone (AB) regeneration because of their same embryonic origin and superior osteogenic potential. However, their molecular processes during osteogenic differentiation remain unclear. The objective of this study was to identify the responsiveness of dental follicle cells (DFCs) and AB marrow-derived mesenchymal stem cells (ABM-MSCs) to recombinant human bone morphogenetic protein-2 (rhBMP-2). These cells expressed vimentin and MSC markers and did not express cytokeratin and hematopoietic stem cell markers and showed multilineage differentiation potential under specific culture conditions. DFCs exhibited higher proliferation and colony-forming unit-fibroblast efficiency than ABM-MSCs; rhBMP-2 induced DFCs to differentiate toward a cementoblast/osteoblast phenotype and ABM-MSCs to differentiate only toward a osteoblast phenotype; and rhBMP-2-induced DFCs exhibited higher osteogenic differentiation potential than ABM-MSCs. These cells adhered, grew, and produced extracellular matrix on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA). During a 14-day culture on nHAC/PLA, the extracellular alkaline phosphatase (ALP) activity of DFCs decreased gradually and that of ABM-MSCs increased gradually; rhBMP-2 enhanced their extracellular ALP activity, intracellular osteocalcin (OCN), and osteopontin (OPN) protein expression; and DFCs exhibited higher extracellular ALP activity and intracellular OCN protein expression than ABM-MSCs. When implanted subcutaneously in severe combined immunodeficient mice for 3 months, DFCs+nHAC/PLA+rhBMP-2 obtained higher percentage of bone formation area, OCN, and cementum attachment protein expression and lower OPN expression than ABM-MSCs+nHAC/PLA+rhBMP-2. These results showed that DFCs possessed superior proliferation and osteogenic differentiation potential in vitro, and formed higher quantity and quality bones in vivo. It suggested that DFCs might exhibit a more sensitive responsiveness to rhBMP-2, so that DFCs enter a relatively mature stage of osteogenic differentiation earlier than ABM-MSCs after rhBMP-2 induction. The findings imply that these dental stem/progenitor cells are alternative sources for AB engineering in regenerative medicine, and developing dental tissue may provide better source for stem/progenitor cells.

Combined Use of Recombinant Human BMP-7 and Osteogenic Media May Have No Ideal Synergistic Effect on Leporine Bone Regeneration of Human Umbilical Cord Mesenchymal Stem Cells Seeded on Nanohydroxyapatite/Collagen/Poly (l-Lactide).

Human umbilical cord mesenchymal stem cells (hUC-MSCs) are a promising alternative source of mesenchymal stem cells (MSCs) that are enormously attractive for clinical use. This study was designed to investigate the effect of recombinant human bone morphogenetic protein-7 (rhBMP-7) and/or osteogenic media (OMD) on bone regeneration of hUC-MSCs seeded on nanohydroxyapatite/collagen/poly(l-lactide) (nHAC/PLA) in a rabbit model. The characteristics of stem cells were analyzed by plastic adherence, cell phenotype, and multilineage differentiation potential. Cell proliferation was examined using cell counting kit-8 assay. Osteogenic differentiation was evaluated by quantitative Ca2+ concentration, PO43- concentration, alkaline phosphatase (ALP) activity, osteocalcin (OCN) secretion, and mineralized matrix formation. Bone regeneration was investigated in jaw bone defect repair in rabbit by microcomputed tomography, fluorescent labeling, and hematoxylin and eosin staining. Except for initial stress response, OMD and OMD + rhBMP-7 inhibited the proliferation of hUC-MSCs seeded on nHAC/PLA; rhBMP-7 inhibited cell proliferation in the nonlogarithmic phase and attenuated the inhibitory effect of OMD on cell proliferation. The inhibitory effects of OMD, rhBMP-7, and OMD + rhBMP-7 on cell proliferation were ranked as OMD > OMD + rhBMP-7 > rhBMP-7. OMD, rhBMP-7, and OMD + rhBMP-7 promoted Ca2+ concentration, PO43- concentration, ALP activity, OCN secretion, and mineralized matrix formation of hUC-MSCs seeded on nHAC/PLA. The promoting effects of OMD, rhBMP-7, and OMD+rhBMP-7 on Ca2+ concentration, PO43- concentration, ALP activity, OCN secretion, and mineralized matrix formation were ranked as rhBMP-7 > OMD > OMD + rhBMP-7, OMD > OMD + rhBMP-7 > rhBMP-7, OMD > rhBMP-7 > OMD + rhBMP-7, rhBMP-7 > OMD + rhBMP-7 > OMD, and OMD > rhBMP-7 > OMD + rhBMP-7, respectively. In rabbit jaw bone defect repair, OMD, rhBMP-7, and OMD + rhBMP-7 enhanced bone regeneration of hUC-MSCs seeded on nHAC/PLA, but the largest bone mineral apposition rate and bone formation were presented in cultures with rhBMP-7. These findings suggested that the combined use of rhBMP-7 and OMD may have no ideal synergistic effect on bone regeneration of hUC-MSCs seeded on nHAC/PLA in rabbit jaw bone defect.

Identification, characterization and microRNA expression profiling of side population cells in human oral squamous cell carcinoma Tca8113 cell lines.

The present study aimed to evaluate the stem cell markers, characteristics and biological functions of cancer stem­like side population (SP) cells in human oral cancer. SP cells were isolated from the human oral squamous cell carcinoma Tca8113 cell line by Hoechst 33342 fluorescence dye and flow cytometry. The colony forming and proliferative capability of SP and non­SP cells were detected using a live­cell analysis system in vitro. The number of cells expressing stem cell markers was compared between SP cells and non­SP cells by flow cytometry. Reverse transcription­quantitative polymerase chain reaction and western blotting were used to detect the mRNA and protein expression levels of stem cell genes, respectively. Differential expression of microRNAs (miRNAs) in SP and non­SP cells was determined by microarray hybridization and an miRNA regulation network was produced. With regard to the proliferation capability, SP cells reached 60.0% confluence after 40 h of growth compared with 35.1% confluence for non­SP cells (P<0.05). The number of colonies in SP cells was 43.1±9.2 compared with 33.0±8.2 of non­SP cells (P<0.05). The aldehyde dehydrogenase­1 (ALDH1)­positive cell number in the SP cells was increased by 10 times compared with the non­SP cells (P<0.01). The mRNA and protein expression levels of ALDH1, SRY­box 2, POU class 5 homeobox 1 and Nanog homeobox in SP cells were significantly higher compared with non­SP cells (P<0.05). Microarray hybridization demonstrated that 21 miRNAs were upregulated and 13 miRNAs were downregulated in SP cells compared with non­SP cells. SP cells in Tca8113 demonstrated greater capability of proliferation and colony formation compared with non­SP cells in vitro. Stem cell markers were overexpressed in SP cells compared with non­SP cells.

Tantalum-incorporated hydroxyapatite coating on titanium implants: its mechanical and in vitro osteogenic properties.

OBJECTIVE: The fabrication of bioactive coatings on metallic implants to enhance osseointegration has become a topic of general interest in orthopedics and dentistry. Hydroxyapatite (HA) coating has been shown to induce bone formation and promote bone-implant integration. Unfortunately, poor mechanical performance has hindered this from becoming a favorable coating material. The majority of present studies have focused in incorporating different elements into HA coatings to improve mechanical properties. In recent years, tantalum (Ta) has received increasing attention due to its excellent biocompatibility and corrosion resistance. The aim of on the present study was to investigate the fabrication and biological performance of Ta-incorporated HA coatings. METHODS: Ta-incorporated HA coatings were fabricated using the plasma spray technique on a titanium substrate, and the surface characteristics and mechanical properties were examined. In addition, the effects of Ta-incorporated HA coatings on the biological behavior of mesenchymal stem cells (BMSCs) were investigated. RESULTS: Ta-incorporated HA coatings with microporous structure had higher roughness and wettability. In addition, the bonding strength of Ta/HA coatings with the substrate was substantially superior to HA coatings. Furthermore, Ta-incorporated HA coatings not only facilitated initial cell adhesion and faster proliferation, but also promoted the osteogenic differentiation of BMSCs. CONCLUSION: These results indicate that the incorporation of Ta could improve mechanical performance and increase the osteogenic activity of HA coatings. The Ta-incorporated HA coating fabricated by plasma spraying is expected to be a promising bio-coating material for metallic implants.

[Minimal invasive microscopic tooth preparation based on endodontic, periodontal and functional health].

Tooth preparation is the primary and core operation technique for dental esthetic restoration treatment, due to its effect of providing restoration space, bonding interfaces and marginal lines for dental rehabilitation after tooth tissue reduction. The concept of microscopic minimal invasive dentistry put forward the issue of conducting high-quality tooth preparation, conserve tooth-structure, protect vital pulp and periodontal tissue simultaneously. This study reviewed the concepts, physiology background, design and minimal invasive microscopic tooth preparation, and in the meantime, individualized strategies and the two core elements of tooth preparation (quantity and shape) are listed.

Brain activation patterns during unilateral premolar occlusion.

OBJECTIVE: To observe the characteristics of brain activation during unilateral premolar occlusion. METHODS: Functional magnetic resonance imaging was collected from 10 healthy volunteers during occlusion of the left first premolar (L1), left second premolar (L2), and right first premolar (R1). The brain activation patterns were analyzed, and the primary sensorimotor cortex, supplementary motor area, insula, thalamus, and prefrontal cortex were chosen as regions of interest. RESULTS: Single premolar occlusion activated the precentral gyrus, postcentral gyrus, cerebellum, thalamus, frontal lobe, hippocampus, cingulate gyrus, and parietal lobe. The brain areas showing activation during single premolar occlusion were similar to those activated by chewing. The activation pattern of L1 was more similar to that of L2 than R1. No significant left and right hemisphere differences in signal intensity were detected within the regions of interest. CONCLUSION: Brain activation patterns from two ipsilateral premolars were more similar than the pattern from a contralateral premolar.

[Comparative study on bilateral temporomandibular joint of patients with unilateral multiple symptoms in cone-beam computed tomography].

PURPOSE: To investigate bilateral temporomandibular joint of patients with unilateral multiple symptoms in cone-beam computed tomography (CBCT) and explore the reference planes that may be different,providing reference for the diagnosis of temporomandibular disorders and comparative study. METHODS: 50 cases with unilateral multiple symptoms（except for cases with unilateral single symptom）were examined by CBCT and the following indexes were observed and analyzed,including horizontal angles of the cross-sectional condyle after the reconstruction in the same patient, joint space, macroaxis diameter of condyle and vertical angles of condyle, which were commonly used at oblique position parallelled to the long axis of condyle, the gradient of articular tubercle and the joint space,which could be obtained at sagittal and oblique position vertical to the long axis of condyle.The data obtained was analyzed by paired t test with SPSS13.0 software package. RESULTS: There was significant difference between the bilateral measured value of joint space when the angle was 60° in sagittal plane (P<0.05).The difference was more significant when the angle was 120° in parallel plane and 90° in sagittal plane (P<0.01). The other measured parameters were not significant different. CONCLUSIONS: For patients with TMD, it is more easily to observe differences between the bilateral measured value of joint space in the sagittal or vertical plane,where the increase of the front joint space can be seen and construction was more significant.

The complete mitochondrial genome of bearded pig, Sus barbatus, and comparative mitochondrial genomics of Cetartiodactyla.

In this study, the complete mitochondrial genome sequence of bearded pig, Sus barbatus, with the total length of 16,480 bp, is determined for the first time. This mitogenome harbors 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and one control region (D-loop). The overall base composition is A (34.80%), C (26.07%), G (13.12%), and T (26.01%), so the slight A-T bias (60.81%) was detected. Most of the genes are distributed on the H-strand, except for the ND6 subunit gene and eight tRNA genes. To obtain the phylogenetic relationship of the Cetartiodactyla, 11 mitochondrial genomes were used for phylogenetic analysis. The mitochondrial genome of S. barbatus presented here will contribute to a better understanding of the population genetics.

Effects of Danggui-Shaoyao-San on the Influence of Spatial Learning and Memory Induced by Experimental Tooth Movement.

BACKGROUND: The pain caused by orthodontic treatment has been considered as tough problems in orthodontic practice. There is substantial literature on pain which has exactly effected on learning and memory; orthodontic tooth movement affected the emotional status has been showed positive outcomes. Danggui-Shaoyao-San (DSS) is a Traditional Chinese Medicine prescription that has been used for pain treatment and analgesic effect for orthodontic pain via inhibiting the activations of neuron and glia. We raised the hypothesis that DSS could restore the impaired abilities of spatial learning and memory via regulating neuron or glia expression in the hippocampus. METHODS: A total of 36 rats were randomly divided into three groups: (1) Sham group (n = 12), rats underwent all the operation procedure except for the placement of orthodontic forces and received saline treatment; (2) experimental tooth movement (ETM) group (n = 12), rats received saline treatment and ETM; (3) DSS + ETM (DETM) group (n = 12), rats received DSS treatment and ETM. All DETM group animals were administered with DSS at a dose of 150 mg/kg. Morris water maze test was evaluated; immunofluorescent histochemistry was used to identify astrocytes activation, and immunofluorescent dendritic spine analysis was used to identify the dendritic spines morphological characteristics expression levels in hippocampus. RESULTS: Maze training sessions during the 5 successive days revealed that ETM significantly deficits in progressive learning in rats, DSS that was given from day 5 prior to ETM enhanced progressive learning. The ETM group rats took longer to cross target quadrant during the probe trial and got less times to cross-platform than DETM group. The spine density in hippocampus in ETM group was significantly decreased compared to the sham group. In addition, thin and mature spine density were decreased too. However, the DSS administration could reverse the dendritic shrinkage and increase the spine density compared to the ETM group. Astrocytes activation showed the opposite trend in hippocampus dentate gyrus (DG). CONCLUSIONS: Treatment with DSS could restore the impaired abilities on ETM-induced decrease of learning and memory behavior. The decreased spines density in the hippocampus and astrocytes activation in DG of hippocampus in the ETM group rats may be related with the decline of the ability of learning and memory. The ability to change the synaptic plasticity in hippocampus after DSS administration may be correlated with the alleviation of impairment of learn and memory after ETM treatment.

Enhancement of periodontal tissue regeneration by transplantation of osteoprotegerin-engineered periodontal ligament stem cells.

INTRODUCTION: The objective of the present study was to evaluate the capacity of a tissue-engineered complex of human osteoprotegerin (hOPG)-transfected periodontal ligament stem cells (PDLSCs) seeding on beta-tricalcium phosphate (ß-TCP) to regenerate alveolar bone defects in New Zealand rabbits. METHODS: PDLSCs were isolated from rabbit periodontal ligament tissues and expanded in vitro to enrich PDLSC numbers, and their proliferative activities and differentiation capability were evaluated under specific induction conditions. Lentiviral vector containing hOPG and enhanced green fluorescent protein (EGFP) was constructed by using Gateway technology and transfected into rabbit PDLSCs. The expression of hOPG was determined with quantitative real-time reverse transcription-polymerase chain reaction and Western blot. The PDLSCs with or without engineered hOPG were seeded on ß-TCP scaffolds prior to transplantation. Morphological characterization of cells and materials was done by scanning electron microscope. Twenty rabbits with alveolar bone defects were randomly allocated into four groups and transplanted with ß-TCP, PDLSCs/ß-TCP, and hOPG-transfected PDLSCs/ß-TCP or were left untreated as a control. Animals were sacrificed 12 weeks after operation for histological observation and histomorphometric analysis. RESULTS: PDLSCs expressed STRO-1 and vementin and favored osteogenesis and adipogenesis in conditioned media. Expressions of hOPG were significantly upregulated after transfection of the lentiviral vector into PDLSCs. PDLSCs attached and spread well on ß-TCP, and there was no significant difference in growth of PDLSCs on ß-TCP between the hOPG transfection group and the non-transfection group. The histological observation and histomorphometric analysis showed that the hOPG-transfected PDLSCs/ß-TCP complex exhibited an earlier mineralization and more bone formation inside the scaffold than control, ß-TCP, and PDLSCs/ß-TCP complexes. Implantation of hOPG-transfected PDLSCs contributed to new bone formation as determined by EGFP gene expression under circularly polarized light microscopy. CONCLUSIONS: The present study demonstrated the feasibility of ß-TCP scaffolds for primary PDLSC culture and expression of hOPG gene in vitro and in vivo, and hOPG-transfected PDLSCs could serve as a potential cell source for periodontal bone regeneration, which may shed light on the potential of systemic hOPG gene therapy in combination with PDLSC tissue engineering as a good candidate in periodontal tissue engineering for alveolar bone regeneration.

Lipofuscin in saliva and plasma and its association with age in healthy adults.

BACKGROUND AND AIM: To compare blood and salivary levels of lipofuscin in healthy adults and to analyze the relationship between the lipofuscin level and the healthy adults' age. METHODS: One hundred and twenty-two healthy volunteers were recruited and divided into three groups according to their age: young (n = 42, 20-44 years old), middle-aged (n = 51, 45-59 years old), and elderly (n = 29, 60-74 years old). One ml saliva and 5 ml whole blood were collected from each person. An ELISA kit was used to measure both the plasma and salivary lipofuscin levels. The differences between the groups were compared with independent-sample t test, and the relationship between the salivary lipofuscin level and the age was assessed with linear regression analysis. RESULTS: The mean ± SD of the lipofuscin level in the saliva and plasma of 122 subjects was 68.93 ± 1.32 and 78.05 ± 1.75 µmol/l, respectively. No gender-dependent differences were observed in either the salivary or the plasma lipofuscin level (saliva: p = 0.443, plasma: p = 0.459). The salivary and plasma lipofuscin levels of the elderly subjects were significantly higher than those of the young (saliva: 80.72 ± 13.53 mmol/l versus 59.12 ± 1.92 mmol/l, p = 0.0003; plasma: 93.31 ± 3.14 mmol/l versus 67.43 ± 2.54 mmol/l, p = 0.0002) and middle-aged (saliva: 80.72 ± 13.53 mmol/l versus 70.31 ± 11.17 mmol/l, p = 0.0004; plasma: 93.31 ± 3.14 mmol/l versus 78.12 ± 2.40 mmol/l, p = 0.0002) subjects. Similarly, the salivary and plasma lipofuscin levels of the middle-aged subjects were significantly higher than those of the young subjects (saliva: 70.31 ± 11.17 mmol/l versus 59.12 ± 1.92 mmol/l, p < 0.0001; plasma: 78.12 ± 2.40 mmol/l versus 67.43 ± 2.54 mmol/l, p = 0.0019). The lipofuscin levels in the saliva and plasma were significantly positively correlated with the subject age (r = 0.551, p = 0.0001; r = 0.528, p < 0.0001). Furthermore, the salivary lipofuscin level and plasma lipofuscin level also were found to have a positive correlation (r = 0.621, p < 0.0001). CONCLUSION: No gender-dependent differences were observed in either the salivary or plasma lipofuscin levels. The salivary and plasma lipofuscin levels were positively correlated, and the age is positively correlated with lipofuscin content in saliva.

Synergistic cytotoxicity of cisplatin and Taxol in overcoming Taxol resistance through the inhibition of LDHA in oral squamous cell carcinoma.

The development of chemoresistance in patients represents a major challenge in cancer treatment. Lactate dehydrogenase-A (LDHA) is one of the principle isoforms of LDH that is expressed in breast tissue, controlling the conversion of pyruvate to lactate and also playing a significant role in the metabolism of glucose. The aim of this study was to identify whether LDHA was involved in oral cancer cell resistance to Taxol and whether the downregulation of LDHA, as a result of cisplatin treatment, may overcome Taxol resistance in human oral squamous cells. The OECM-1 oral epidermal carcinoma cell line was used, which has been widely used as a model of oral cancer in previous studies. The role of LDHA in Taxol and cisplatin resistance were investigated and the synergistic cytotoxicity of cisplatin and/or Taxol in oral squamous cells was analyzed. Cell viability was analyzed by MTT assay, LDHA expression was analyzed by western blot analysis and siRNA tranfection was performed to knock down LDHA expression. The present study results showed that decreased levels of LDHA were responsible for the resistance of oral cancer cells to cisplatin (CDDP). CDDP treatments downregulated LDHA expression, and lower levels of LDHA were detected in the CDDP-resistant oral cancer cells compared with the CDDP-sensitive cells. By contrast, the Taxol-resistant cancer cells showed elevated LDHA expression levels. In addition, small interfering RNA-knockdown of LDHA sensitized the cells to Taxol, but desensitized them to CDDP treatment, while exogenous expression of LDHA sensitized the cells to CDDP, but desensitized them to Taxol. The present study also revealed the synergistic cytotoxicity of CDDP and Taxol for killing oral cancer cells through the inhibition of LDHA. This study highlights LDHA as a novel therapeutic target for overcoming Taxol resistance in oral cancer patients using the combined treatments of Taxol and CDDP.

Subcellular location and photodynamic therapeutic effect of chlorin e6 in the human tongue squamous cell cancer Tca8113 cell line.

The present study aimed to investigate the distribution and photodynamic therapeutic effect of chlorin e6 (Ce6) in the human tongue squamous cell carcinoma Tca8113 cell line in vitro. The distribution of Ce6 in the Tca8113 cells was observed in situ combined with mitochondrial and lysosomal fluorescent probes. Next, 630-nm semiconductor laser irradiation was performed. The MTS colorimetric method was used to determine cell survival. Annexin V fluorescein isothiocyanate/propidium iodide (PI) double staining was used to detect early apoptosis following photodynamic therapy (PDT). The flow cytometer was used to analyze the DNA content subsequent to PI-staining. It was observed that Ce6 could combine with the cellular membrane following 30 min of incubation with the Tca8113 cells. As the length of incubation increased, Ce6 gradually entered the cells in a particular distribution and reached saturation by 3 h. Co-localization analysis demonstrated that Ce6 was more likely to be present in the mitochondria than in the lysosomes. The cells incubated with 5 µg/ml Ce6 for 24 h exhibited a low toxicity of 5%, however, following light irradiation, Ce6-PDT was able to kill the Tca8113 cells in vitro. The cell toxicity was positively correlated with Ce6 concentration and light dose, therefore, the effect of Ce6 was concentration/dose-dependent (P<0.01). The lower Ce6 concentrations and light doses could significantly induce apoptosis in the Tca8113 cells, while higher doses increased necrosis/percentage of dead cells. In summary, Ce6 saturated the Tca8113 cells following 3 h of incubation. Furthermore, Ce6-PDT effectively killed the cultured Tca8113 cells in vitro at a safe concentration. At a low concentration and light dose, Ce6 is more likely to induce cell apoptosis via the mitochondria than the lysosomes.

The effect of calcium phosphate composite scaffolds on the osteogenic differentiation of rabbit dental pulp stem cells.

The objective of this study is to compare the effects of the two calcium phosphate composite scaffolds on the attachment, proliferation, and osteogenic differentiation of rabbit dental pulp stem cells (DPSCs). One nano-hydroxyapatite/collagen/poly (l-lactide) (nHAC/PLA), imitating the composition and the micro-structure characteristics of the natural bone, was made by Beijing Allgens Medical Science & Technology Co., Ltd. (China). The other beta-tricalcium phosphate (ß-TCP), being fully interoperability globular pore structure, was provided by Shanghai Bio-lu Biomaterials Co, Ltd. (China). We compared the absorption water rate and the protein adsorption rate of two scaffolds and the characterization of DPSCs cultured on the culture plate and both scaffolds under osteogenic differentiation media (ODM) treatment. The constructs were then implanted subcutaneously into the back of severely combined immunodeficient (SCID) mice for 8 and 12 weeks to compare their bone formation capacity. The results showed that the ODM-treated DPSCs expressed osteocalcin (OCN), bone sialoprotein (BSP), type I collagen (COLI) and osteopontin (OPN) by immunofluorescence staining. Positive alkaline phosphatase (ALP) staining, calcium deposition and calcium nodules were also observed on the ODM-treated DPSCs. The absorption water rate and protein adsorption rate of nHAC/PLA was significantly higher than ß-TCP. The initial attachment of DPSCs seeded onto nHAC/PLA was significantly higher than that onto ß-TCP; and the proliferation rate of the cells was also significantly higher than that of ß-TCP on 1, 3, and 7 days of cell culture. The ALP activity, calcium/phosphorus content and mineral formation of DPSCs + ß-TCP were significantly higher than DPSCs + nHAC/LA. When implanted into the back of SCID mice, nHAC/PLA alone had no new bone formation, newly formed mature bone and osteoid were only observed in ß-TCP alone, DPSCs + nHAC/PLA and DPSCs + ß-TCP, and this three groups displayed increased bone formation over the 12-week period. The percentage of total bone formation area had no difference between DPSCs + ß-TCP and DPSCs + nHAC/PLA at each time point, but the percentage of mature bone formation area of DPSCs + ß-TCP was significantly higher than that of DPSCs + nHAC/PLA. Our results demonstrated that the DPSCs on nHAC/PLA had a better proliferation, and that the DPSCs on ß-TCP had a more mineralization in vitro, much more newly formed mature bones in vivo were presented in DPSCs + ß-TCP group. These findings have provided a further knowledge that scaffold architecture has different influence on the attachment, proliferation and differentiation of cells. This study may provide insight into the clinical periodontal bone tissue repair with DPSCs + ß-TCP construct.

[Observing the effect of high glucose on proliferation of bone marrow stromal stem cells through Wnt/Β-catenin pathway].

OBJECTIVE: To explore the effect of high glucose on proliferation of bone marrow stromal stem cells through Wnt/Β-catenin pathway. METHODS: Bone marrow stormal cells were obtained from the mandible of Wistar rats and stimulated with different concentrations of glucose (5.5 and 16.5 mmol/L). Cell proliferation was evaluated with methyl thiazolyl tetrazolium assay (1, 3, 5, and 7 d)and cell cycle analysis by flow cytometry (5 d). Β-catenin and cyclin D1 protein levels were determined by Western blot. The mRNA expression of lymphoid enhancer binding factor-1 (LEF-1) and cyclin D1 were tested by real-time polymerase chain reaction. RESULTS: The results of methyl thiazolyl tetrazolium assay indicated that the optical density values of two different concentrations of the glucose had no statistical difference on day 1 (P=0.700). On days 3, 5, and 7, the optical density values of the 16.5 mmol/L group were significantly lower than those in the 5.5 mmol/L group (P=0.006, P=0.002, and P=0.003). Cell cycle analysis indicated that high glucose concentration could reduced the progression from phase G1 to S, and the proliferation index values of the 16.5 mmol/L group were significantly lower than those of the 5.5 mmol/L group (P=0.014). The Β-catenin and cyclin D1 levels were lower in the 16.5 mmol/L group when compared with the 5.5 mmol/L group. High glucose condition also reduced the mRNA expressions of LEF-1 and cyclin D1. CONCLUSION: High glucose can inhibit the proliferation of bone marrow stormal cells by suppressing the expressions of Β-catenin, LEF-1, and cyclin D1 in the Wnt/Β-catenin pathway.

[Effect of the peri-implantitis on the biological function of osteoblasts obtained from the mandibles].

OBJECTIVE: To study the effect of peri-implantitis inflammatory microenvironment on the biological function of jaw bone osteoblasts. METHODS: Primary mandible osteoblasts from peri-implantitis and normal tissue were isolated and cultured. Third-generation purified osteoblasts were identified and detected. The proliferative activity of osteoblasts was evaluated through MTT assay. Osteocalcin (OCN), Runx2, and collagen I (Col I) mRNA levels were examined by real-time quantitative polymerase chain reaction. OCN protein levels were determined by Western blot. RESULTS: : After 4 d of culture, the proliferative activity of osteoblasts from peri-implantitis became lower than that of normal tissue ( P <0.05). After 7 d of culture, OCN, Runx2, and Col I mRNA expression decreased ( P <0.05). The OCN protein levels also decreased ( P <0.05). CONCLUSION: Peri-implantitis inflammatory microenvironment can decrease the proliferation and differentiation activity of mandible osteoblasts.

Palatal rugae for the construction of forensic identification / Rugas palatinas en identificación forense

This study aims to explore the role of palatal rugae pattern in forensic identification by coding of palatal rugae characteristics, and to construct a forensic identification system for oral palatal rugae. One hundred models were included in this study for a systemic coding of palatal rugae pattern based on the shape, quantity, location, and distribution of palatal rugae. Among the involved 100 models, palatal rugae types varied among individuals and palatal rugae pattern was different between men and women, even between two sides in the same individual. Palatal rugae pattern can be used for forensic identification of oral soft tissue and this study proposes a new means for the identification by coding of palatal rugae pattern based on the shape, quantity, location and distribution.

Este estudio tuvo como objetivo explorar el patrón de rugas palatinas en la identificación forense mediante la codificación de las características de éstas. Además se elaboró un sistema de identificación forense. Fueron incluidos cien modelos para una codificación sistemática del patrón de rugas palatinas basado en su forma, cantidad, ubicación y distribución. En todos los modelos se identificó una variación entre los individuos y en el patrón entre hombres y mujeres. El patrón de rugas palatinas puede ser utilizado para la identificación forense de tejido blando oral y este estudio propone un método nuevo para codificarlas.