Human ß-defensins are correlated with the immune infiltration and regulated by vitamin D3 in periodontitis.

OBJECTIVE: Exploring the correlation between human ß-defensins (HBDs) and immune infiltration in periodontitis, and whether it is regulated by vitamin D3 . BACKGROUND: The human body produces essential antimicrobial peptides called HBDs, which are associated with periodontitis. There is a strong link between periodontal tissue destruction and the immune cell infiltration. Moreover, vitamin D3 has been reported to regulate the expression of immune cell chemokines. However, the relationship between vitamin D3 , HBDs, and immune infiltration in periodontitis remains to be investigated. METHODS: The Gene Expression Omnibus database was accessed to obtain transcriptomic information of gingival samples taken from periodontitis patients. The expression value of HBD-2 and HBD-3 was calculated. Additionally, using the online program ImmuCellAl, 10 immune cells were scored for immune infiltration in the high-HBDs-expression group and the low-HBDs-expression group, separately. After that, transcriptome sequencing was done based on human gingival fibroblasts that had received vitamin D3 treatment. Furthermore, hGFs were treated by vitamin D3 , tumor necrosis factor-α (TNF-α), and Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). The expressions of HBD-2, HBD-3, interleukin-8 (IL-8), and monocyte chemoattractant protein-1 (MCP-1) were detected. To seek the potential mechanism, CYP27A1 siRNA was employed to reduce the expression of CYP27A1, and nuclear factor-gene binding protein 65 (NF-κB p65) was examined. RESULTS: In GSE10334, the expressions of HBD-2 and HBD-3 were down-regulated in periodontitis group. Meanwhile, monocyte, macrophage, and CD4_T cell were less infiltrated in low-HBD-2-expression group, while less Gamma-delta T-cell infiltration was found in low-HBD-3-expression group. Transcriptome sequencing found that 21 genes were significantly expressed, of which the function was enriched in response to bacterial origin and TNF signal pathway. Vitamin D3 could significantly up-regulate the expression of HBD-2 and HBD-3, which could be controlled by knocking down CYP27A1 mRNA expression. With prolonged vitamin D3 stimulation, the expression of HBD-2 and HBD-3 increased. TNF-α/Pg-LPS could significantly increase the expression of HBD-2, HBD-3, IL-8, MCP-1, and p65, all of which were reduced by vitamin D3 . CONCLUSION: HBDs are correlated with immune infiltration in periodontitis. Vitamin D3 inhibits the expression of HBDs and chemokines induced by TNF-α/Pg-LPS, possibly through NF-κB pathway, in human gingival fibroblasts.

Immunogenic Cell Death in Hematological Malignancy Therapy.

Although the curative effect of hematological malignancies has been improved in recent years, relapse or drug resistance of hematological malignancies will eventually recur. Furthermore, the microenvironment disorder is an important mechanism in the pathogenesis of hematological malignancies. Immunogenic cell death (ICD) is a unique mechanism of regulated cell death (RCD) that triggers an intact antigen-specific adaptive immune response by firing a set of danger signals or damage-associated molecular patterns (DAMPs), which is an immunotherapeutic modality with the potential for the treatment of hematological malignancies. This review summarizes the existing knowledge about the induction of ICD in hematological malignancies and the current research on combining ICD inducers with other treatment strategies for hematological malignancies.

Smart One-for-All Agent with Adaptive Functions for Improving Photoacoustic /Fluorescence Imaging-Guided Photodynamic Immunotherapy.

Multifunctional phototheranostics that integrate several diagnostic and therapeutic strategies into one platform hold great promise for precision medicine. However, it is really difficult for one molecule to possess multimodality optical imaging and therapy properties that all functions are in the optimized mode because the absorbed photoenergy is fixed. Herein, a smart one-for-all nanoagent that the photophysical energy transformation processes can be facilely tuned by external light stimuli is developed for precise multifunctional image-guided therapy. A dithienylethene-based molecule is designed and synthesized because it has two light-switchable forms. In the ring-closed form, most of the absorbed energy dissipates via nonradiative thermal deactivation for photoacoustic (PA) imaging. In the ring-open form, the molecule possesses obvious aggregation-induced emission features with excellent fluorescence and photodynamic therapy properties. In vivo experiments demonstrate that preoperative PA and fluorescence imaging help to delineate tumors in a high-contrast manner, and intraoperative fluorescence imaging is able to sensitively detect tiny residual tumors. Furthermore, the nanoagent can induce immunogenic cell death to elicit antitumor immunity and significantly suppress solid tumors. This work develops a smart one-for-all agent that the photophysical energy transformation and related phototheranostic properties can be optimized by light-driven structure switch, which is promising for multifunctional biomedical applications.

Platelet-rich fibrin/anorganic bovine bone mineral complex as grafting materials in endodontic microsurgery with a large lesion size: study protocol for a randomised controlled trial.

INTRODUCTION: Endodontic microsurgery is a treatment of last resort for preserving natural teeth. According to radiographic evaluation, the percentage of complete healing after endodontic microsurgery is only 74.3%. The use of regenerative techniques in endodontic microsurgery for large lesions (>10 mm diameter) is therefore recommended. The most frequently used bone graft in endodontic microsurgery is anorganic bovine bone mineral (ABBM) but this only has an osteoconductive effect. Thus, when platelet-rich fibrin (PRF), a reservoir of growth factors, is used together with ABBM, it increases the regenerative effect. This study is devoted to comparing the clinical outcomes of PRF with/without ABBM as grafting biomaterials in endodontic microsurgery cases with large lesion size to provide some valuable reference data for dentists. METHODS AND ANALYSIS: Sixteen patients who are in need of endodontic microsurgery will be recruited. The patients will be randomly assigned to one of two groups: an experimental group, treated with PRF/ABBM complex and collagen membrane, and a control group, treated with ABBM and collagen membrane. Clinical examination including percussion, mobility testing and presence/absence of sinus will be recorded at 7 days, and at 3, 6 and 12 months after endodontic microsurgery. A Visual Analogue Scale will be used by the patients to evaluate pain at 1, 3 and 7 days after endodontic microsurgery. Routine paralleling radiographs will be obtained before and at 3, 6 and 12 months follow-up after endodontic microsurgery. Cone-beam CT (CBCT) scans will be obtained at the 12-month follow-up. Bone formation will be evaluated according to CBCT and paralleling radiographs. The study execute time including follow-ups last from 1 June 2021 to 31 December 2024. ETHICS AND DISSEMINATION: This study received approval from the Ethics Committee of Peking University School and Hospital of Stomatology. The results will be disseminated through scientific journals. TRIAL REGISTRATION NUMBER: Research data will be registered with the International Clinical Trials Registry Platform (ICTRP), ID: ChiCTR2100046684.

Effects of DDR1 on migration and adhesion of periodontal ligament cells and the underlying mechanism.

BACKGROUND AND OBJECTIVE: As one of the widely expressed cell surface receptors binding to collagen, the most abundant component of the extracellular matrix (ECM), knowledge of the expression, functions, and mechanisms underlying the role of discoidin domain receptor 1 (DDR1) in human periodontal ligament cells (hPDLCs) is incomplete. This study determined the expression of DDR1 in hPDLCs and the effect of DDR1 upon migration and adhesion to hPDLCs, as well as the related regulatory mechanisms. MATERIALS AND METHODS: The expression of DDR1 and the DDR1 isoforms in hPDLCs from six donors were tested. The migratory ability (horizontal and vertical) and adhesive capacity of hPDLCs with or without specific knockdown of DDR1 were evaluated. After treatment with MEK-ERK1/2 inhibitors (PD98059 and U0126) with or without RNAi, the migratory and adhesive capacity of hPDLCs were re-tested. Western blotting was performed to verify p-MEK1/2 and p-ERK1/2, the key factors of the MEK-ERK1/2 signaling pathways. RESULTS: DDR1 was detected in hPDLCs in the mRNA and protein level; DDR1b was the dominant isoform. Knockdown of DDR1 almost halved the migratory capacity and significantly downregulated the adhesive capacity of hPDLCs. The use of MEK-ERK1/2 inhibitors caused declined migratory and adhesive capacity of hPDLCs as well. After DDR1 was knocked down, the expression of p-MEK and p-ERK protein declined significantly while total MEK and ERK showed no obvious change, which means the ratio of p-MEK/MEK and p-ERK/ERK was markedly reduced. CONCLUSIONS: DDR1 plays an important role in the migration and adhesion of hPDLCs and might be regulated via the MEK-ERK1/2 signaling pathway.

Treatment of periodontal intrabony defects using bovine porous bone mineral and guided tissue regeneration with/without platelet-rich fibrin: A randomized controlled clinical trial.

BACKGROUND: To investigate the regenerative effect of adjunctive use of guided tissue regeneration (GTR), bovine porous bone mineral (BPBM), and platelet-rich fibrin (PRF) in intrabony defects. METHODS: Fourteen participants were enrolled, and for each patient their left and right two sides were randomized to the test group or control group. Only the worst intrabony defect on each side was analyzed. The test group received GTR, BPBM, and PRF, whereas the control group received only GTR and BPBM. The PRF used in the trial was fluid PRF, which combined with the BPBM to form a BPBM-PRF complex. The patients were followed up by clinical and radiographic evaluation for 24 months after surgery. RESULTS: Probing depth (PD) in the test group was significantly less than that in the control group at 12 and 24 months after surgery, and the mean difference was ≈ 0.5 to 0.7 mm. Clinical attachment level (CAL) gain in the test group was ≈ 0.9 mm higher than that in the control group at 6 months after surgery, and the difference reached 1.0 to 1.1 mm 12 and 24 months after surgery. None of the other clinical or radiographic parameters differed significantly between the two groups at any time-point after the surgery. CONCLUSION: Compared with GTR and BPBM, the combination of GTR and BPBM-PRF complex is more effective clinically, and results in better clinical outcomes.

Expression of vitamin D 1α-hydroxylase in human gingival fibroblasts in vivo.

BACKGROUND: Vitamin D 1α-hydroxylase CYP27B1 is the key factor in the vitamin D pathway. Previously, we analyzed the expression of CYP27B1 in human gingival fibroblasts in vitro. In the present study, we analyzed the gingival expression of CYP27B1 in vivo. METHODS: Forty-two patients with periodontitis Stage IV Grade C and 33 controls were recruited. All patients with periodontitis had unsalvageable teeth and part of the wall of the periodontal pocket was resected and obtained after tooth extraction. All controls needed crown-lengthening surgery, and samples of gingiva resected during surgery were also harvested. All the individuals' gingivae were used for immunohistochemistry and immunofluorescence. In addition, gingivae from seventeen subjects of the diseased group and twelve subjects of the control group were analyzed by real-time PCR. RESULTS: Expression of CYP27B1 was detected both in gingival epithelia and in gingival connective tissues, and the expression in connective tissues colocalized with vimentin, indicating that CYP27B1 protein is expressed in gingival fibroblasts. The expression of CYP27B1 mRNA in gingival connective tissues and the CYP27B1 staining scores in gingival fibroblasts in the diseased group were significantly higher than those in the control group. CONCLUSIONS: Expression of CYP27B1 in human gingival tissues was detected, not only in the fibroblasts of gingival connective tissues, but also in the gingival epithelial cells, and might be positively correlated with periodontal inflammation.

The Non-N6-Methyladenosine Epitranscriptome Patterns and Characteristics of Tumor Microenvironment Infiltration and Mesenchymal Transition in Glioblastoma.

Background: An increasing number of RNA modification types other than N6-methyladenosine (m6A) modification have been detected. Nonetheless, the probable functions of RNA modifications beyond m6A in the tumor microenvironment (TME), mesenchymal (MES) transition, immunotherapy, and drug sensitivity remain unclear. Methods: We analyzed the characteristics of 32 non-m6A RNA modification regulators in 539 glioblastoma (GBM) patients and the TME cell infiltration and MES transition patterns. Using principal component analysis, a non-m6A epitranscriptome regulator score (RM score) model was established. We estimated the association between RM score and clinical characteristics, TME status, GBM subtypes, and drug and immunotherapy response. Results: Three definite non-m6A RNA modification patterns associated with diverse biological pathways and clinical characteristics were identified. The high RM score group was characterized by a poor prognosis, enhanced immune infiltration, and MES subtype. Further analysis indicated that the high RM score group had a lower tumor mutation burden as well as a weaker response to immunotherapy. The higher RM score group may benefit more from drugs targeting the EGFR and WNT signaling pathways. Conclusion: Our study exposed the potential relationship of non-m6A RNA modification regulators with clinical features, TME status, and GBM subtype and clarified its therapeutic value.

Extending the vitamin D pathway to vitamin D3 and CYP27A1 in periodontal ligament cells.

BACKGROUND: In periodontal connective tissue cells, the vitamin D pathway has been elucidated, and vitamin D3 in the main storage form, 25-hydroxy vitamin D3 (25[OH]D3 ), and the functional form, 1,25-dihydroxy vitamin D3 (1,25[OH]2 D3 ), have been found to induce the expression of human cationic antimicrobial protein (hCAP-18)/LL-37. Moreover, synergistic effects between Toll-like receptor agonists and 25(OH)D3 have been reported. This research aimed at extending the vitamin D pathway to vitamin D3 and CYP27A1 in human periodontal ligament cells (hPDLCs) to further explore its function in periodontal inflammatory reaction. METHODS: Vitamin D3 was used to stimulate hPDLCs in the presence or absence of Porphyromonas gingivalis lipopolysaccharide (Pg-LPS). Conversely, CYP27A1 RNA interference was performed to further validate the findings. The mRNA expression of hCAP-18 was determined with real-time polymerase chain reaction. Monocyte chemotactic protein-1 (MCP-1) and interleukin-8 (IL-8) were also detected. The cell supernatant levels of LL-37 were detected with enzyme-linked immunosorbent assay. RESULTS: Vitamin D3 significantly enhanced the generation of hCAP-18/LL-37. A combination of Pg-LPS and vitamin D3 significantly promoted hCAP-18/LL-37 expression. When the expression of CYP27A1 was knocked down with RNA interference, the induction of hCAP-18/LL-37 expression was significantly inhibited. Therefore, the mRNA levels of MCP-1 and IL-8 in hPDLCs were significantly decreased through the vitamin D pathway. CONCLUSION: The vitamin D pathway from vitamin D3 to hCAP-18/LL-37 exists in hPDLCs, and CYP27A1 might be involved in periodontal immune defense.

Modified minimally invasive surgical technique plus Bio-Oss Collagen for regenerative therapy of isolated interdental intrabony defects: study protocol for a randomised controlled trial.

INTRODUCTION: Periodontal regeneration surgery has been widely used to deal with intrabony defects. Modified minimally invasive surgical technique (M-MIST) is designed to deal with isolated interdental intrabony defects, and has achieved satisfactory periodontal regenerative effect. Bio-Oss Collagen, as a bioactive material, has been applied for periodontal regeneration. It is similar to human cancellous bone, with the ability to promote bone formation; furthermore, it has exceptional plasticity and spatial stability. The combination of different materials and techniques has become a research hotspot in recent years. By combining the superiority of regeneration technology and materials, better regenerative effect can be achieved. This study will search for differences between M-MIST combined with Bio-Oss Collagen, and M-MIST alone in regeneration therapy for intrabony defects. METHODS AND ANALYSIS: The present research is designed as a two-group parallel randomised controlled trial. The total number of patients is 40. The patients will be randomly assigned to two groups, with 20 participants in each group, for further periodontal regenerative surgery. Test group: M-MIST plus Bio-Oss Collagen. CONTROL GROUP: M-MIST. After 12 months, the measurement indices will be recorded; these will include clinical attachment gain and radiographical intrabony defect depth change as the primary results, and secondary outcomes of full-mouth plaque scores, probing depth, full-mouth bleeding scores, gingival recession, mobility, gingival papilla height and Visual Analogue Scale. The paired samples t-test will be applied to detect any difference between baseline and 1-year registrations. A general linear model will be performed to study the relationship between the secondary and the primary outcome. ETHICS AND DISSEMINATION: The present research has received approval from the Ethics Committee of Peking University School and Hospital of Stomatology (PKUSSIRB-202053002). Data of the present research will be registered with the International Clinical Trials Registry Platform. Additionally, we will disseminate the results through scientific dental journals. TRIAL REGISTRATION NUMBER: ChiCTR-2000030851. PROTOCOL VERSION: Protocol Version 4, 14 July 2020.

Preliminary investigation on the molecular mechanisms underlying the correlation between VDR-FokI genotype and periodontitis.

BACKGROUND: The only polymorphism that could change the protein structure in vitamin D receptor (VDR) is the FokI polymorphism (rs2228570). The FF genotype has the strongest transcriptional activity of VDR and is correlated with higher susceptibility to periodontitis. To reveal the possible molecular mechanisms for the correlation preliminarily, the influence of VDR-FokI genotype on the expression of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa B ligand (RANKL) in human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) was investigated in this study. METHODS: hGFs and hPDLCs from 15 donors (five FF, seven Ff, and three ff) were treated with 1,25OH2 D3 , with or without the specific knockdown of VDR using siRNA. The mRNA and protein expression of OPG and RANKL were detected using real-time PCR and enzyme-linked immunosorbent assay, respectively. RESULTS: Both in hGFs and hPDLCs, 1,25OH2 D3 could significantly induce the mRNA and protein expression of RANKL, and FF genotype had significantly higher induction than the other genotypes, however, neither 1,25OH2 D3 nor VDR-FokI had significant influence on the OPG expression. As a result, the RANKL/OPG ratio was significantly elevated under 1,25OH2 D3 stimulation and FF genotype had the most remarkable elevation. When VDR was knocked down, all the differences among the three genotypes disappeared. CONCLUSION: The strongest transcriptional activity of FF genotype might contribute to the strongest enhancement of RANKL expression and RANKL/OPG ratio in hGFs and hPDLCs stimulated by 1,25OH2 D3 , which might help to reveal the mechanisms of the correlation between FF genotype and susceptibility to periodontitis.

Preliminary investigation of the vitamin D pathway in periodontal connective tissue cells.

BACKGROUND: The vitamin D pathway, from toll-like receptor activation to human cationic antimicrobial protein (hCAP-18/LL-37) generation, has been identified in monocytes and keratinocytes. This study aimed to investigate the vitamin D pathway in human gingival fibroblasts (hGFs) and human periodontal ligament cells (hPDLCs) and to provide preliminary evidence of its role in periodontal immune defense. METHODS: Primary cultures of hGFs and hPDLCs were stimulated with 1,25-dihydroxy vitamin D3 and 25-hydroxy vitamin D3 , with or without Porphyromonas gingivalis lipopolysaccharide. CYP27B1 RNA interference and vitamin D receptor (VDR) antagonism were also used for reverse proof. The mRNA expression of hCAP-18/LL-37, VDR, interleukin (IL)-6, IL-8, and monocyte chemotactic protein-1 were detected using real-time polymerase chain reaction. The LL-37 concentrations were measured using enzyme-linked immunosorbent assay. RESULTS: In hGFs and hPDLCs, 25-hydroxy vitamin D3 and 1,25-dihydroxy vitamin D3 induced hCAP-18/LL-37 expression, which was further increased by Porphyromonas gingivalis lipopolysaccharide. If the function of CYP27B1 or VDR was blocked, the induction was significantly weakened. IL-8 and monocyte chemotactic protein-1 mRNA expression could be suppressed by the vitamin D pathway. CONCLUSION: These findings suggest that the vitamin D pathway exists in hGFs and hPDLCs and plays an important role in immune defense in periodontal soft tissues.

Janus Nanoparticles for Improved Dentin Bonding.

The amphiphilic monomer 2-hydroxyethyl methacrylate (HEMA) is widely used in dental adhesives as a priming component, especially for dentin bonding. It behaves as a compatibilizer between hydrophilic and hydrophobic components and stabilizes the multicomponent adhesive system. However, there are several drawbacks associated with using HEMA, such as water retention within the adhesive layer, hydrolysis in oral environments, and cytotoxicity. These drawbacks lead to the failure of tooth restoration and represent a heavy medical burden. Thus, it is imperative to find a new compatibilizer to substitute for HEMA. Because of their superior compatibilization capabilities as functional solid surfactants, amphiphilic Janus particles are chosen as candidates for an alternative to HEMA in dental adhesives. Reactive amphiphilic Janus nanoparticles are synthesized by selectively etching and modifying at the interface of a Pickering emulsion. This approach could be extended to the synthesis of a series of other Janus nanoparticles. The Janus nanoparticles were verified to be better for the reduction of the phase separation and stabilization of dentin adhesives than HEMA. It is also demonstrated that these reactive Janus nanoparticles can strongly enhance the dentin bonding interface without cytotoxicity. It is clearly illustrated by this study that Janus nanoparticles may be promising materials to substitute for HEMA in dental adhesives.

Influence of rs2228570 on Transcriptional Activation by the Vitamin D Receptor in Human Gingival Fibroblasts and Periodontal Ligament Cells.

BACKGROUND: rs2228570 is the only known single nucleotide polymorphism of the vitamin D receptor (VDR) that alters the protein structure. VDRs can be distinguished using the restriction endonuclease FokI and accordingly divided into three genotypes: FF, Ff, and ff. Influence of rs2228570 on transcriptional activation by VDRs in human gingival fibroblasts (hGFs) and periodontal ligament cells (hPDLCs) is investigated in this study. METHODS: From 15 donors, hGFs and hPDLCs were cultured, genomic DNA was extracted, and genotypes were determined using the polymerase chain reaction (PCR)-restriction fragment length polymorphism method. Cells were stimulated with calcitriol with or without VDR antagonist ZK159222 or osteogenic induction. Alkaline phosphatase, osteocalcin, and VDR messenger RNA (mRNA) expression were detected using real-time PCR. Alkaline phosphatase and osteocalcin protein expression were detected by enzyme activity assays with p-nitrophenyl phosphate substrate and enzyme-linked immunosorbent assay, respectively. RESULTS: Among the 15 donor cell cultures, the number of FF, ff, and Ff genotypes were 5, 3, and 7, respectively. There were no significant differences in expression of alkaline phosphatase or osteocalcin among the three genotypes in hGFs. However, after stimulation with calcitriol, alkaline phosphatase and osteocalcin mRNA levels in FF-hPDLCs were significantly higher than in other hPDLCs genotypes, as was osteocalcin protein expression. Furthermore, when ZK159222 was included, this difference disappeared, and when osteogenic induction was performed, alkaline phosphatase and osteocalcin mRNA and protein levels were higher in FF-hPDLCs than in the other hPDLCs genotypes. CONCLUSION: The FF-VDR genotype is associated with the most remarkable upregulation of alkaline phosphatase and osteocalcin in hPDLCs.

Upregulated Leptin in Periodontitis Promotes Inflammatory Cytokine Expression in Periodontal Ligament Cells.

BACKGROUND: Imbalance or disruption in the expression of inflammatory mediators contributes greatly to the breakdown of the periodontal supporting tissues. Leptin, through binding to its receptor (obesity-related leptin and leptin receptor [OBR]), has potent effects on immunity and inflammation. However, to date, researchers only indicated a role of leptin in periodontitis. No direct or valid evidence exists about how leptin and its receptor are regulated by local inflammation, what effects they have, and the underlying mechanisms. METHODS: Experimental periodontitis was induced by ligation of mandibular second molars in beagle dogs. The expression of leptin, OBR, and interleukin (IL)-1ß was examined by immunohistochemistry. Meanwhile, recombinant human IL-1ß was used to stimulate human periodontal ligament cells (hPDLCs) in vitro, and mRNA and protein levels of leptin were measured using real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA), respectively. Then, mRNA and protein levels of IL-6 and IL-8 were measured using real-time PCR and ELISA, after stimulation with various concentrations of leptin, knocking down all or only the long form of OBR (OBRb) by small interfering RNA and incubation with multiple intracellular signaling pathway inhibitors, respectively. RESULTS: Leptin and OBR increased substantially in inflammatory periodontal tissues, which correlated well with the extent of inflammatory infiltration, and was a result of the upregulation in resident cells themselves. A high dose of leptin could induce the expression of mRNA and protein of IL-6 and IL-8 in hPDLCs through binding with OBRb and activating different intracellular signaling pathways. CONCLUSION: Upregulated leptin and OBR in periodontitis stimulated proinflammatory cytokine expression in PDL cells to additionally promote local inflammation.

[Characteristic of sample banks isolated from EDTA-blood by sedimentation method].

OBJECTIVE: To assess the characteristics of establishing the different sample banks of plasma, leukocytes and DNA by sedimentation method of isolating from ethylene diamine tetraacetic acid(EDTA)-blood and to clarify the sedimentation method of leukocyte isolation and plasma volume by comparative data and recommended procedures for applicability. METHODS: In the study, 29 EDTA-bloods were obtained, the total amounts of leukocytes and the percentage of neutrophile granulocytes, and lymphocytes in the EDTA-blood detected as a control group and then assigned equally into 4 EP tubes with 1 mL EDTA-blood per tube as 4 test groups, then the 4 tubes were placed with the EDTA-blood at room temperature and the plasma layers were isolated at 0.5, 1, 2 and 3 h, receptively. The total amount of leukocytes and the percentage of neutrophile granulocytes, and lymphocytes were detected by automated hematology analyzer at the clinical laboratory. The volume of the plasma was also measured at the same time. RESULTS: The plasma volume at 0.5 h [(241.72 ± 101.52)µL] was substantially lower than those at 1 h[(317.24 ± 97.50)µL], at 2 h[(371.03 ± 91.66)µL], and at 3 h [(408.97 ± 97.43)µL] , P < 0.05. The plasma volume at 1 h was substantially lower than those at 2 h and 3 h (P < 0.05). The total amount of leukocytes in the plasma layer at 0.5 h (2.50 × 10(6) ± 1.48 × 10(6)) group was substantially higher than the amount of 2 or 3 h groups respectively (1.47 × 10(6) ± 7.19 × 105,1.21 × 10(6) ± 7.41 × 105), P < 0.05. Significant difference was not found between 0.5 h group and 1 h group (2.29 × 10(6)± 1.17 × 10(6)), P > 0.05. The total amount of leukocytes in the plasma layer in 1 h group was substantially higher than that in 2 h and 3 h groups (P < 0.05). There was no significant difference between 3 h group and 2 h group (P > 0.05). The total amount of leukocytes in the plasma layer of the 4 test groups was substantially lower than that in the control group (P < 0.05). The percentage of neutrophile granulocytes (54.14% ± 11.65%) in the plasma layer in 0.5 h group was substantially higher than those in 1 h, 2 h and 3 h groups (46.66% ± 12.70%,39.17% ± 12.33%,43.25% ± 14.54%), P < 0.05, respectively, which was the substantially lower than that in the control group (60.53% ± 8.46%), P < 0.05. The average value of the percentage of neutrophile granulocytes in the plasma layer in 1 h group was substantially higher than that in 2 h group (P < 0.05). There was no significant different between 3 h group and both 1 h, 2 h groups (P > 0.05). The mean percentage of lymphocytes in the plasma layer in 0.5 h group (35.09% ± 10.84%) was substantially lower than those in the plasma layer in 1 h, 2 h and 3 h groups, respectively ( 41.48% ± 12.20%, 47.96% ± 12.27%, 45.50% ± 13.71%), which was significant higher than that in the control group(30.98% ± 7.33%), P < 0.05. The average value of the percentage of lymphocytes in the plasma layer in 1 h group was substantially higher than those in the control group and 0.5 h group, but was substantially lower than those in 2 h and 3 h groups (P < 0.05). The average value of percentage of lymphocytes in the plasma layer in 2 h group was substantially higher than those in the control group, 0.5 h and 1 h groups (P < 0.05). There was no significant difference between 2 h and 3 h groups (P > 0.05). CONCLUSION: The best period of time in obtaining leukocytes is 0.5-1 h sedimentation of EDTA-blood. Both the plasma layer and leukocytes can be separated and obtained at the same time from the same sample by the sedimentation method of EDTA-blood. The sedimentation of EDTA-blood has the least interference of both chemical and physical factors, as well as a ready operation, which can establish the plasma, leukocytes and DNA sample banks for various aspects of research.

Leptin and its receptor expression in dental and periodontal tissues of primates.

Leptin and its receptor (OBR) have attracted much attention since their discovery. They have been reported to play central roles in energy balance, the immune-inflammatory response and bone metabolism. Evidence indicates that leptin and OBR are associated with inflammatory diseases of dental and periodontal tissues. The first step for establishing this is to determine the expression of leptin and OBR in these tissues. Our study is the first to examine systematically the expression of leptin and OBR in dental and periodontal tissues of monkeys (Macaca fascicularis) by immunohistochemistry and in primary cultured cells, isolated from human dental and periodontal tissues, by reverse transcription plus the polymerase chain reaction and immunocytochemistry. Our results show that leptin and OBR are constitutively expressed and widely distributed in dental and periodontal tissues of primates. Their immunoreaction is especially strong in junctional epithelium, a unique front-line defense around teeth and in mineralizing areas of the dental pulp and periodontal ligament. The expression of the long and also functional form of OBR (OBRb) indicates that leptin has a direct effect on these cells. Thus, we can reasonably infer that leptin and OBR exert effects on defense, mineralization and angiogenesis in dental and periodontal tissues of primates.

Activity of 25-hydroxylase in human gingival fibroblasts and periodontal ligament cells.

BACKGROUND: We previously demonstrated that 25-hydroxyvitamin D(3) concentrations in gingival crevicular fluid are 300 times higher than those in the plasma of patients with aggressive periodontitis. Here we explored whether 25-hydroxyvitamin D(3) can be synthesized by periodontal soft tissue cells. We also investigated which of the two main kinds of hydroxylases, CYP27A1 and CYP2R1, is the key 25-hydroxylase in periodontal soft tissue cells. METHODOLOGY/PRINCIPAL FINDINGS: Primary cultures of human gingival fibroblasts and periodontal ligament cells from 5 individual donors were established. CYP27A1 mRNA, CYP2R1 mRNA and CYP27A1 protein were detected in human gingival fibroblasts and periodontal ligament cells, whereas CYP2R1 protein was not. After incubation with the 25-hydroxylase substrate vitamin D(3), human gingival fibroblasts and periodontal ligament cells generated detectable 25-hydroxyvitamin D(3) that resulted in the production of 1α,25-dihydroxyvitamin D(3). Specific knockdown of CYP27A1 in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 25-hydroxyvitamin D(3) and 1α,25-dihydroxyvitamin D(3) production. Knockdown of CYP2R1 did not significantly influence 25-hydroxyvitamin D(3) synthesis. Sodium butyrate did not influence significantly CYP27A1 mRNA expression; however, interleukin-1ß and Porphyromonas gingivalis lipopolysaccharide strongly induced CYP27A1 mRNA expression in human gingival fibroblasts and periodontal ligament cells. CONCLUSIONS: The activity of 25-hydroxylase was verified in human gingival fibroblasts and periodontal ligament cells, and CYP27A1 was identified as the key 25-hydroxylase in these cells.

Characterization of the autocrine/paracrine function of vitamin D in human gingival fibroblasts and periodontal ligament cells.

BACKGROUND: We previously demonstrated that 25-hydroxyvitamin D(3), the precursor of 1α,25-dihydroxyvitamin D(3), is abundant around periodontal soft tissues. Here we investigate whether 25-hydroxyvitamin D(3) is converted to 1α,25-dihydroxyvitamin D(3) in periodontal soft tissue cells and explore the possibility of an autocrine/paracrine function of 1α,25-dihydroxyvitamin D(3) in periodontal soft tissue cells. METHODOLOGY/PRINCIPAL FINDINGS: We established primary cultures of human gingival fibroblasts and human periodontal ligament cells from 5 individual donors. We demonstrated that 1α-hydroxylase was expressed in human gingival fibroblasts and periodontal ligament cells, as was cubilin. After incubation with the 1α-hydroxylase substrate 25-hydroxyvitamin D(3), human gingival fibroblasts and periodontal ligament cells generated detectable 1α,25-dihydroxyvitamin D(3) that resulted in an up-regulation of CYP24A1 and RANKL mRNA. A specific knockdown of 1α-hydroxylase in human gingival fibroblasts and periodontal ligament cells using siRNA resulted in a significant reduction in both 1α,25-dihydroxyvitamin D(3) production and mRNA expression of CYP24A1 and RANKL. The classical renal regulators of 1α-hydroxylase (parathyroid hormone, calcium and 1α,25-dihydroxyvitamin D(3)) and Porphyromonas gingivalis lipopolysaccharide did not influence 1α-hydroxylase expression significantly, however, interleukin-1ß and sodium butyrate strongly induced 1α-hydroxylase expression in human gingival fibroblasts and periodontal ligament cells. CONCLUSIONS/SIGNIFICANCE: In this study, the expression, activity and functionality of 1α-hydroxylase were detected in human gingival fibroblasts and periodontal ligament cells, raising the possibility that vitamin D acts in an autocrine/paracrine manner in these cells.

[Effects of emdogain on human periodontal ligament cells in vitro].

OBJECTIVE: To investigate the effects of emdogain, enamel matrix derivative (EMD), on the proliferation, cell cycle, mineralization and ultrastructure of human periodontal ligament (PDL) cells in vitro. METHODS: The influence of cell growth on PDL cells was evaluated by Cell Counting Kit-8 (CCK-8) in the presence and absence of emdogain, after 1, 3, and 5 d of culture. DNA synthesis and ultrastructure of PDL cells were observed by flow cytometry(FCM) and transmission electron microscopy (TEM) in the presence and absence of emdogain after 3 d of culture. The increasing of osteogenic capacity was verified by the expression changes of osteogenic differentiation markers of bone sialoprotein (BSP) and osteopontin (OPN) in emdogain-treated PDL cells by immunohistochemicl staining. RESULTS: Incubation of PDL cells with emdogain after 3 d significantly stimulated cell growth and DNA synthesis. Emdogain enhanced the osteogenic potential of PDL cells by high expression of osteogenic differentiation markers of BSP and OPN. CONCLUSION: The data indicate that Emdogain enhances cell proliferation and promotes differentiation of PDL cells, which contributes to periodontal tissue regeneration .