Genome-Wide Screening of Differentially Expressed Genes and their Potential Associations with Aging Dental Pulp Stem Cells.

BACKGROUND: Dental pulp stem cells (DPSCs) refer to a type of stem cells, which is characterized by great differentiation potential and is easy to obtain. DPSCs are able to be employed for treating immune diseases and tissue regeneration. However, the differentiation ability exhibited by aging DPSCs is reduced, thereby limiting the application. As speculated by the microarray analysis, different expressions of miRNAs might be involved in DPSC senescence, whereas comprehensive transcriptome level detection has been rare. OBJECTIVE AND METHODS: To gain insights into the molecular mechanisms involved, RNA-sequencing, pathway enrichment and Gene Ontology Analysis were conducted on aging and young DPSCs. RESULTS: In this study, the differences in long non-coding RNA (lncRNA) and messenger RNA (mRNA expressions) of the aging and young DPSCs were demonstrated, and the vital factors and the relevant pathways were speculated. On the whole, 18950 mRNAs and 21854 lncRNAs were detected, among which 14 mRNAs and 7 lncRNAs were differentially expressed. Furthermore, hsa-miR-6724-5p may be a vital node in the aging process of DPSCs, and its target genes was involved in the dopaminergic synapse. CONCLUSION: In brief, the aging of DPSCs was significantly dependent of differentially expressed genes (DEGs) which is related to dopaminergic synapse. However, the specific function and internal relationship of the DEGs should be verified in depth.

Antibacterial and Antioxidant Effects of Magnesium Alloy on Titanium Dental Implants.

OBJECTIVES: In this study, a new type of dental implant by covering the surface of the titanium (Ti) implant with zinc-magnesium (Zn-Mg) alloy was designed, to study the antibacterial and antioxidant effects of Mg alloy on titanium (Ti) implants in oral implant restoration. METHODS: Human gingival fibroblasts (HGFs), S. sanguinis, and F. nucleatum bacteria were used to detect the bioactivity and antibacterial properties of Mg alloy-coated Ti implants. In addition, B6/J mice implanted with different materials were used to further detect their antibacterial and antioxidant properties. RESULTS: The results showed that Mg alloy could better promote the adhesion and proliferation and improve the alkaline phosphatase (ALP) activity of HGFs, which contributed to better improved stability of implant osseointegration. In addition, Mg alloy could better inhibit the proliferation of S. sanguinis, while no significant difference was found in the proliferation of F. nucleatum between the two implants. In the mouse model, the peripheral inflammatory reaction and oxidative stress of the Mg alloy implant were significantly lower than those of the Ti alloy implant. CONCLUSIONS: Zn-Mg alloy-coated Ti implants could better inhibit the growth of Gram-positive bacteria in the oral cavity, inhibit oxidative stress, and facilitate the proliferation activity of HGFs and the potential of osteoblast differentiation, thus, better increasing the stability of implant osseointegration.

Cytotoxicity, Oxidative Stress, and Autophagy Effects of Tantalum Nanoparticles on MC3T3-E1 Mouse Osteoblasts.

As a bone implant material, porous tantalum (Ta) has better corrosion resistance and more suitable elastic modulus than titanium. Surface nanomodification can accelerate the integration of Ta implants with bone tissue, which has broad application prospects in the field of dental implantology. Due to mechanical stress and load wear, nanoscale Ta fragments are inevitably exfoliated from the implant surface and brought into direct contact with osteoblasts surrounding the implant. These wear fragments may affect the biological characteristics of osteoblasts and thus the stability of implants. To date, the interaction of nanoscale Ta fragments with osteoblasts has not been clearly investigated. In the current study, we used the mouse osteoblast cell line MC3T3-E1 to explore the effects of Ta nanoparticles (Ta-NPs) on the cytotoxicity, oxidative stress and autophagy of osteoblasts. We found that a low concentration (12.5 µg/mL) of Ta-NPs can promote the proliferation of osteoblasts, while the Ta-NPs began to induce a decrease in cell viability at concentrations ≥25 µg/mL. Increased cell mortality, reactive oxygen species (ROS) production and decreased mitochondrial membrane potential (MMP) occurred in a dose-dependent manner after Ta-NP treatment. Moreover, with Ta-NP stimulation, the ratio of LC3-II/LC3-I increased, and the level of p62 protein was reduced. However, the degradation of p62 was not continuously increased when the concentration of Ta-NPs was ≥25 µg/mL. These results indicate that Ta-NPs induced osteoblast damage via oxidative stress. Autophagy activation may be a key factor in the cellular response to Ta-NP toxicity and could have an important impact on determining the survival or death of osteoblasts.

The effects of separating inferior alveolar neurovascular bundles on osteogenesis of tissue-engineered bone and vascularization.

AIM: To evaluate the effects of autologous blood vessels and nerves on vascularization. METHODS: A dog model of tissue-engineered bone vascularization was established by constructing inferior alveolar neurovascular bundles through the mandibular canal. Sixteen 12-month-old healthy beagles were randomly divided into two groups (n=8). Group A retained inferior alveolar neurovascular bundles, and Group B retained inferior alveolar nerves. Bone marrow mesenchymal stem cells were injected into ß-tricalcium phosphate to prepare internal tissue-engineered bone scaffold. A personalized titanium mesh was then prepared by rapid prototyping and fixed by external titanium scaffold. Two dogs in each group were sacrificed on the 30th, 45th, 60th and 90th postoperative days respectively. The bone was visually examined, scanned by CT, and subjected to HE staining, immunohistochemical staining, vascular casting and PCR to detect the changes in osteogenesis and vascularization. RESULTS: The two groups had similar outcomes in regard to osteogenesis and vascularization (P>0.05): both showed remarkable regenerative capacities. CONCLUSIONS: The model of tissue-engineered bone vascularization is potentially applicable in clinical practice to allow satisfactory osteogenesis and vascularization.

Variations of sulfhydryl compounds of saliva and plasma in healthy population of different ages / 中华口腔医学杂志

<p><b>OBJECTIVE</b>To analyze the effect of age and gender on sulfhydryl compounds content in saliva and plasma in healthy population and to study the relationship between sulfhydryl compounds content of saliva and plasma to provide a basis for clinical examination of saliva sulfhydryl compounds.</p><p><b>METHODS</b>Sulfhydryl compounds content of saliva and plasma were measured in 306 healthy adults from the Department of Clinical Laboratory of Health Management lnstitute of General Hospital of Chinese PLA (151 female and 155 male) who were divided into young group (20-44 years old, n = 106, 48 female and 58 male), middle-aged group (45-59 years old, n = 109, 63 female and 46 male) and elderly group (60-79 years old, n = 91, 40 female and 51 male).</p><p><b>RESULTS</b>Sulfhydryl compounds content in saliva and plasma in 306 healthy adults were (123±27) and (427±124) µmol/L respectively. Sulfhydryl compounds content in saliva and plasma were significantly decreased as age increased (both P < 0.01). Significant differences of sulfhydryl compounds content of saliva and plasma among the young group, middle-aged group and elderly group were found (P < 0.01). No sex difference was observed in saliva sulfhydryl compounds content (P = 0.451), however the sex difference was significant in plasma sulfhydryl compounds content (P = 0.006). There was a significantly positive correlation between sulfhydryl compounds content in saliva and plasma (r = 0.5050, P < 0.01).</p><p><b>CONCLUSIONS</b>Saliva sulfhydryl compounds content can roughly reflect plasma sulfhydryl compounds content. Saliva sulfhydryl compounds test is a promising biological index of aging which could be an alternative of plasma test.</p>

Danggui-shaoyao-san, a traditional Chinese medicine prescription, alleviates the orthodontic pain and inhibits neuronal and microglia activation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The pain caused by orthodontic treatment has been considered as tough problems in orthodontic practice. Danggui-shaoyao-san (DSS) is a traditional Chinese medicine (TCM) prescription which has long been used for pain treatment and possesses antioxidative, cognitive enhancing and antidepressant effects. We raise the hypothesis that DSS exerts analgesic effect for orthodontic pain via inhibiting the activations of neuron and microglia.</p><p><b>METHODS</b>DSS was given twice a day from day 5 prior to experimental tooth movement (ETM). Directed face grooming and vacuous chewing movements (VCM) were evaluated. Immunofluorescent histochemistry and Western blot analysis were used to quantify the Iba-1 (microglia activation) and Fos (neuronal activation) expression levels in the trigeminal spinal nucleus caudalis (Vc).</p><p><b>RESULTS</b>ETM significantly increased directed face grooming and VCM which reached the peak at post-operative day (POD) 1 and gradually decreased to the baseline at POD 7. However, a drastic peak increase of Fos expression in Vc was observed at 4 hours and gradually decreased to baseline at POD 7; while the increased Iba-1 level reached the peak at POD 1 and gradually decreased to baseline at POD 7. Furthermore, pre-treatment with DSS significantly attenuated the ETM induced directed face grooming and VCM as well as the Fos and Iba-1 levels at POD 1.</p><p><b>CONCLUSION</b>Treatment with DSS had significant analgesic effects on ETM-induced pain, which was accompanied with inhibition of both neuronal and microglial activation.</p>

Effect of the peri-implantitis on the biological function of osteoblasts obtained from the mandibles / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To study the effect of peri-implantitis inflammatory microenvironment on the biological function of jaw bone osteoblasts.</p><p><b>METHODS</b>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.</p><p><b>RESULTS</b>: 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).</p><p><b>CONCLUSION</b>Peri-implantitis inflammatory microenvironment can decrease the proliferation and differentiation activity of mandible osteoblasts.</p>

High glucose inhibits osteogenic differentiation through the BMP signaling pathway in bone mesenchymal stem cells in mice.

Patients with diabetes tend to have an increased risk of osteoporosis that may be related to hyperglycemia. In vitro evidence has shown that high glucose can affect the proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs). Tissue regeneration depends mainly on MSCs. However, the exact mechanisms involved in high glucose-induced bone loss remain unknown. In this study, we investigated the effects of high glucose on the proliferation and osteogenic differentiation of mice bone MSCs (BMSCs) and determined the specific mechanism of bone morphogenetic protein 2 (BMP-2) in the osteogenic differentiation of mice BMSCs in a high-glucose microenvironment. High glucose (< 25 mM) promoted cell growth but suppressed mineralization. The intracellular BMP-2 level in BMSCs cultured in a high-glucose microenvironment was significantly decreased and suppressed activation of the BMP signaling pathway. Consequently, expression of the osteogenic markers Runx2, alkaline phosphatase, and osteocalcin were decreased. Meanwhile, supplementation with ectogenic BMP-2 reversed the cell osteogenic differentiation and osteogenic marker down-regulation under high glucose. Our data indicate that BMP-2 plays an important role in regulating the osteogenic differentiation of BMSCs in a high-glucose microenvironment. Thus, it is possible that agents modifying this pathway could be used by BMSCs to promote bone regeneration in high-glucose microenvironments.

Determination of pazufloxacin concentration in saliva, gingival crevicular fluid and serum of healthy adults using high-performance liquid chromatography / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish a rapid and sensitive high-performance liquid chromatography (HPLC) method for detecting pazufloxacin concentrations in the saliva, gingival crevicular fluid and serum of healthy adults.</p><p><b>METHODS</b>Samples of saliva, gingival crevicular fluid and serum were obtained from healthy adults receiving intravenous infusion of pazufloxacin. The concentrations of pazufloxacin in the samples were quantified by HPLC equipped with a reversed-phase column (Agilent Zorbax SB-C18 5 µm, 250 mm×4.6 mm). The mobile phase for pazufloxacin was a mixture of acetonitrile and 0.5% phosphoric acid containing 1% triethylamine (155:850), and 20 µl of the resulting solution was injected into the HPLC system at a flow rate of 1.0 ml/min. The detection wavelength was set at 245 nm. The samples were first deproteinized by precipitation with methanol followed by supernatant drying; the residue was reconstituted with the mobile phase and centrifuged, and the supernatants were directly injected into the HPLC system.</p><p><b>RESULTS</b>Pazufloxacin in the samples were totally separated without interference by any endogenous substances. The calibration curves showed a good linear regression (r>0.999). The detection limit was 10 ng/ml with within-day and between-day coefficients of variation performance all below 5% and recovery rates all above 91%.</p><p><b>CONCLUSION</b>HPLC is both sensitive and selective for quantification of pazufloxacin in saliva, gingival crevicular fluid and serum.</p>

Sustained local delivery of insulin for potential improvement of peri-implant bone formation in diabetes.

Dental implantation is an effective standard treatment modality to restore missing teeth and maxillofacial defects. However, in diabetics there is an increased risk for implant failure due to impaired peri-implant osseous healing. Early topical insulin treatment was recently shown to normalize diabetic bone healing by rectifying impairments in osteoblastic activities. In this study, insulin/poly(lactic-co-glycolic acid) (PLGA) microspheres were prepared by a double-emulsion solvent evaporation method. Microspheres were then incorporated in fibrin gel to develop a local drug delivery system for diabetic patients requiring implant treatment. In vitro release of insulin from fibrin gel loaded with these microspheres was assessed, and sustained prolonged insulin release over 21 days ascertained. To assess the bioactivity of released insulin and determine whether slow release might improve impaired diabetic bone formation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), alkaline phosphatase (ALP) activity, mineralized nodule formation, and ELISA (enzyme-linked immunosorbent assay) assays were performed. The insulin released from the drug delivery system stimulated cell growth in previously inhibited cells, and ameliorated the impaired bone-forming ability of human MG-63 cells under high glucose conditions. Fibrin gel loaded with insulin/PLGA microspheres shows potential for improving peri-implant bone formation in diabetic patients.

Expression of glucose transporter-1 and insulin receptor alpha1 in osteoblast obtained from diabetic rats' mandibles / 华西口腔医学杂志

<p><b>OBJECTIVE</b>To explore the expression of glucose transporter (GLUT)-1 and insulin receptor (IR) alpha1 in osteoblast obtained from diabetic rats' mandibles.</p><p><b>METHODS</b>The expression of GLUT-1 and IR alpha1 of diabetic and control groups were measured by reverse transcription polymerase chain reaction, Western blot and immunohistochemistry stain.</p><p><b>RESULTS</b>The mRNA expressions of GLUT-1 and IR alpha1 of diabetic group were significantly higher than control group (P<0.05). The protein expressions of GLUT-1 and IR alpha1 were similar to the mRNA expressions.</p><p><b>CONCLUSION</b>Osteoblasts obtained from diabetic rats' mandibles keep the adaptation changes to hyperglycemia and hypoinsulinemia, which may contribute to their dysfunction.</p>

Influence of ADAM28 on biological characteristics of human dental follicle cells.

OBJECTIVES: The aim of this study was to investigate the effects of a disintegrin and metalloproteinase 28 (ADAM28) on the biological characteristics of human dental follicle cells (HDFCs) and possible action mechanism. METHODS: Eukaryotic expression plasmid containing ADAM28 coding region and ADAM28 antisense oligodeoxynucleotides (AS-ODN) with FITC labelling were constructed and synthesised by gene clone and recombination. Then we respectively transfected them into HDFCs by Lipofectamine 2000 system and detected their effects on proliferation, differentiation and apoptosis of HDFCs by MTT assay, cell cycle detection, ALP activity and Annexin V-FITC/PI analysis. Finally we observed the effects of ADAM28 AS-ODN on HDFCs expressing extracellular matrix (ECM) proteins by immunocytochemical staining. RESULTS: ADAM28 eukaryotic plasmid was constructed and identified successfully, and could be correctly translated and expressed in HDFCs, furthermore overexpression of ADAM28 promoted the HDFCs proliferation and inhibited specific differentiation of HDFCs, while inhibition of ADAM28 exerted the opposite effects and induced apoptosis. Moreover ADAM28 could significantly inhibit the secretion of OPN and type III collagen of HDFCs. CONCLUSIONS: ADAM28 might actively participate in the network regulation which associates HDFCs proliferation, differentiation, apoptosis with matrix mineralisation during tooth development by interacting with multiple signal molecules.

Experimental Study of Vascular Tissue Engineering / 中国康复理论与实践

@#Objective To obtain the experimental data of vascular tissue engineering.MethodsThe vascular endothelial cells (VEC) and vascular smooth muscle cells (VSMCs) were acquired and cultured, and then seeded on vascular tissue engineering materials. The porous gelatin-chitosan scaffold with VSMCs was subcutaneously implanted, followed by the observation of the cell growth ten days later.ResultsThe two kinds of cells were successfully cultured and their morpholoical and immunohistochemical characteristics were consistent with vascular endothelial and VSMCs respectively. The VSMCs could grow extensively on the scaffold after the in vivo implantation. The scaffold were wrapped by the fibrous tissue ten days later after the in vitro implantation of VSMCs. The seed cells grew in the scaffold, and the vessel cavity seen in the center of the scaffold, was quite different from the normal vessel structure.ConclusionIt is feasible to implant the VSMCs with fibrin gels into the living body. The vessels reconstructed, though different from the normal structure, is similar to the embryo of the vessels.

Elementary study on construction of engineered blood vessel using chitosan-fibrin gel as scaffold / 中国康复理论与实践

@#ObjectiveTo investigate the feasibility of construction of engineered blood vessel using chitosan tube and fibrin gel as scaffold.MethodsVascular endothelial cells and smooth muscle cells were harvested from aortas of a rat, respectively. After expansion in vitro, vascular endothelial cells were seeded onto the inner surface of chitosan tube and smooth muscle cells mixed with fibrin gel seeded onto outer surface of the scaffold to construct engineered blood vessels. Inverted microscope, immunohistochemical staining and scanning electronic microscope were used to evaluate the construct.ResultsVascular endothelial cells formed monolayer and covered the inner surface of chitosan tube. Smooth muscle cells survived in the fibrin gel and grew in a 3-dimensional manner. ConclusionChitosan-fibrin gel may be potentially used as scaffold of engineered blood vessels.