A Biodegradable Nano-Drug Delivery Platform for Co-Delivery of Minocycline and Chitosan to Achieve Efficient and Safe Non-Surgical Periodontitis Therapy.

INTRODUCTION: Mesoporous silica nanoparticles (MSN) are widely used as ideal nanovehicles for the delivery of chemotherapeutic drugs. However, the balance between high anti-periodontitis activity and low biotoxicity has been challenging to maintain in most relevant studies owing to the slow degradation of silica in living organisms. METHOD: In this study, -responsive hydroxyapatite (HAP) was doped into the MSN skeleton, and the chemotherapeutic drug minocycline hydrochloride (MH) was loaded into the pores of MSN, forming a negatively charged drug delivery system. Cationic chitosan (COS) is a biodegradable material with high antibacterial performance and good biosafety. In this study, COS was immobilized on the surface of the drug-loaded particles through stable charge interaction to construct a composite drug delivery system (MH@MSNion@COS). RESULTS: In vitro and cellular experiments demonstrated effective degradation of the nanocarrier system and synchronized controlled release of the drug. Notably, compared with single MH administration, this system, in which MH and COS jointly regulated the expression levels of periodontitis- associated inflammatory factors (TNF-α, IL-6, IL-1ß, and iNOS), better inhibited the progress of periodontitis and induced tissue regeneration without showing significant toxic side effects in cells. CONCLUSION: This system provides a promising strategy for the design of intelligent, efficient, and safe anti-periodontitis drug delivery systems.

mRNA and long non-coding RNA expression profiling of human periodontal ligament cells under tension loading.

OBJECTIVE: This study explored the expression profiles of messenger RNAs (mRNAs) and long non-coding RNAs (lncRNAs) in human periodontal ligament (PDL) cells subjected to tensile loading. METHODS: PDL cells were isolated from the teeth of five healthy individuals, cultured and then exposed to tensile loading. RNA sequencing was performed to explore the mRNA and lncRNA expression profiles with or without tensile loading. Differential expression, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted to reveal enriched biological functions and signal transduction pathways. Quantitative polymerase chain reaction (qPCR) was performed to validate the expression of specific mRNAs and lncRNAs associated with the enriched pathways. RESULTS: Tensile loading significantly enhanced the osteogenic potential of PDL cells. Overall, 1438 mRNAs (860 up- and 578 down-regulated) and 195 lncRNAs (107 up- and 88 down-regulated) were differentially expressed (adjusted P-value <0.05) in the tensile loading group versus the control group. GO and KEGG analyses of the differentially expressed genes indicated significant enrichment in osteogenesis-related biological processes and intracellular signal transduction pathways (e.g. the PI3K-Akt pathway), respectively. The qPCR analysis validated the expression levels of five selected mRNAs (EGFR, FGF5, VEGFA, HIF1A, and FOXO1) and three selected lncRNAs (CYTOR, MIR22HG, and SNHG3). LIMITATION: Further studies are warranted to validate the mechanisms regulating tension-induced bone remodelling in PDL cells and potential regulation by the identified lncRNAs. CONCLUSION: The notably altered mRNA and lncRNA expression profiles in PDL cells under tensile loading enhance our mechanistic understanding of tension-induced osteogenesis.

Bidirectional ephrinB2­EphB4 signaling regulates the osteogenic differentiation of canine periodontal ligament stem cells.

The aim of the present study was to evaluate the effect of ephrinB2 gene­transfected canine periodontal ligament stem cells (cPDLSCs) on the regulation of osteogenic differentiation. cPDLSCs were transfected with a transgenic null­control green fluorescent protein (GFP) vector (termed Vector­cPDLSCs) or with NFNB2 GFP­Blasticidin (termed EfnB2­cPDLSCs). Subsequently, the osteogenic differentiation of Vector­cPDLSCs and EfnB2­cPDLSCs was assessed by reverse transcription­quantitative polymerase chain reaction (RT­qPCR), alkaline phosphatase (ALP) assay and Alizarin Red S staining. The migratory abilities of cPDLSCs, Vector­cPDLSCs and EfnB2­cPDLSCs were also assessed. Following osteogenic induction of Vector­cPDLSCs and EfnB2­cPDLSCs, the protein expression levels of collagen I, Runt­related transcription factor 2, osteocalcin, ephrin type­B receptor 4 (EphB4), phospho­EphB4, ephrinB2 and phospho­ephrinB2 were analyzed by western blot assays. Following gene transfection, the RT­qPCR and western blotting results revealed that the mRNA and protein expression levels of ephrinB2, respectively, were significantly increased in EfnB2­cPDLSCs compared with that in Vector­cPDLSCs (P<0.05). ALP and Alizarin Red S staining assays revealed increased ALP activity and mineralization nodules, respectively, in EfnB2­cPDLSCs. Cell proliferation and migration assays revealed that EfnB2­cPDLSCs exhibited enhanced proliferation and migration compared with Vector­cPDLSCs (P<0.05). In conclusion, the findings of the current study indicated that ephrinB2 gene­modified cPDLSCs exhibited enhanced osteogenic differentiation, with the ephrinB2 reverse signaling and EphB4 forward signaling pathways serving a key role in this process. Furthermore, ephrinB2 gene modification was observed to promote the migration and proliferation of cPDLSCs.

Lipopolysaccharide inhibits osteogenic differentiation of periodontal ligament stem cells partially through toll-like receptor 4-mediated ephrinB2 downregulation.

OBJECTIVES: This study aimed to investigate the possible crosstalk between LPS/toll-like receptor 4 (TLR4) and ephrinB2 signaling in mediating osteogenic differentiation of PDLSCs. MATERIALS AND METHODS: Human periodontal ligament stem cells (hPDLSCs) were harvested and treated with different concentrations of LPS under osteogenic induction. qPCR, alkaline phosphatase (ALP) staining, and Alizarin Red S staining were performed to assess osteogenic gene expression, ALP activity, and mineralized nodule formation. EphrinB2 mRNA and protein expressions after LPS treatment were also determined. To explore the role of ephrinB2 in LPS-impaired osteogenic differentiation of hPDLSCs, hPDLSCs were stimulated with ephrinB2-Fc or transfected with ephrinB2 lentivirus, and then, the osteogenic differentiation capacity was evaluated. RESULTS: LPS inhibited osteogenic differentiation of hPDLSCs and downregulated ephrinB2 expression in hPDLSCs during osteogenic differentiation. Blockage of TLR4 partially reversed LPS-induced decrease in ephrinB2 expression. EphrinB2-Fc promoted mineralized nodule formation and increased the expression of ALP, osteocalcin (OCN), and bone morphogenetic protein 2 (BMP2) in hPDLSCs. EphrinB2-overexpressing hPDLSCs treated with LPS expressed higher ALP and BMP2 mRNA and higher ALP activity and showed more mineralized nodule formation, when compared with wide-type hPDLSCs treated with LPS. CONCLUSIONS: Our data suggested that LPS decreased the osteogenic differentiation capacity of hPDLSCs partially through downregulation of ephrinB2 expression via LPS/TLR4 signaling. Upregulation of ephrinB2 partially reversed the impaired osteogenic potential of hPDLSCs induced by LPS. CLINICAL RELEVANCE: Our results provided a new insight of mechanism underling LPS-mediated osteogenic differentiation inhibition of PDLSCs and clarified a potential target for the management of periodontitis.

EphrinB2 overexpression enhances osteogenic differentiation of dental pulp stem cells partially through ephrinB2-mediated reverse signaling.

BACKGROUND: Alveolar bone loss is a frequent occurrence. Dental pulp stem cells (DPSCs) which have invasive accessibility and high osteogenic potential is a promising source for cell-based bone regeneration. EphrinB2 is involved in bone homeostasis and osteogenesis. The aim of this study was to investigate the effect and mechanism of ephrinB2 overexpression on osteogenic differentiation of DPSCs and bone defect repair. METHODS: EphrinB2 expression was analyzed during osteogenic induction of human DPSCs (hDPSCs). Endogenous ephrinB2 expression in hDPSCs was then upregulated using EfnB2 lentiviral vectors. The effect of ephrinB2 overexpression on osteogenic differentiation capacity of hDPSCs was investigated in vitro, and activation of ephrinB2-EphB4 bidirectional signaling in ephrinB2-overexpressing hDPSCs was detected. In vivo, a canine alveolar bone defect model was established and canine DPSCs (cDPSCs) were cultured, characterized, EfnB2-tranfected, and combined with a PuraMatrix scaffold. Micro-CT analysis was performed to evaluate the therapeutic effect of ephrinB2-overexpressing cDPSCs on bone defect repair. RESULTS: EphrinB2 was upregulated after osteogenic induction of hDPSCs. EphrinB2 overexpression enhanced osteogenic differentiation capacity of hDPSCs in vitro. Moreover, p-ephrinB2 instead of p-EphB4 was upregulated by ephrinB2 overexpression, and activation of ephrinB2-mediated reverse signaling promoted osteogenic differentiation of hDPSCs. In a canine bone defect model, ephrinB2 overexpression in cDPSCs significantly improved trabecular bone volume per tissue volume (BV/TV) and trabecular thickness, as demonstrated by radiographic analysis. CONCLUSIONS: EphrinB2 overexpression enhanced osteogenic potential of DPSCs partially via upregulation of ephrinB2-mediated reverse signaling and effectively promoted alveolar bone defect repair.

Overexpression of ephrinB2 in stem cells from apical papilla accelerates angiogenesis.

OBJECTIVES: We aimed to accelerate angiogenesis in pulp regeneration by modulating ephrinB2 expression in stem cells from apical papilla (SCAPs). MATERIALS AND METHODS: Stem cells from apical papilla were transducted with ephrinB2-lentiviral expression vector (ephrinB2-SCAPs) in experimental group and green fluorescent protein (GFP-SCAPs) in control group. The transduction efficiency was confirmed by real-time PCR and Western blot assays. MTT assay was performed to detect the proliferative capacity of SCAPs after transduction. In vitro Matrigel assay and in vivo Matrigel plug assay were carried out to evaluate the angiogenic capacity. RESULTS: Results showed that ephrinB2-SCAPs had significantly higher ephrinB2 expression than GFP-SCAPs. EphrinB2-SCAPs upregulated vascular endothelial growth factor (VEGF) secretion under hypoxia. In vitro Matrigel assay demonstrated that human umbilical vein endothelial cells (HUVECs) cocultured with ephrinB2-SCAPs under hypoxia formed vascular-like structures earlier than GFP-SCAPs. Animal experiments confirmed that SCAPs co-transplanted with HUVECs enabled to generate greater amount of blood vessels than SCAPs alone. EphrinB2-SCAPs produced increased number of blood vessels with references to GFP-SCAPs, and those co-transplanted with HUVECs generated vessels with larger and functional tubule volumes. CONCLUSIONS: Regulating ephrinB2 expression in SCAPs may act as a new avenue for enhancing angiogenesis in dental pulp regeneration.

EphrinB2 regulates osteogenic differentiation of periodontal ligament stem cells and alveolar bone defect regeneration in beagles.

EphrinB2, a membrane protein regulating bone homeostasis, has been demonstrated to induce osteogenic gene expression in periodontal ligament fibroblasts. The aim of this study was to explore the effects of ephrinB2 on osteogenic differentiation of periodontal ligament stem cells and on alveolar bone regeneration in vivo. We assessed the osteogenic gene expression and osteogenic differentiation potential of ephrinB2-modified human and canine periodontal ligament stem cells, in which ephrinB2 expression was upregulated via lentiviral vector transduction. EphrinB2-modified canine periodontal ligament stem cells combined with PuraMatrix were delivered to critical-sized alveolar bone defects in beagles to evaluate bone regeneration. Results showed that ephrinB2 overexpression enhanced osteogenic gene transcription and mineral deposition in both human and canine periodontal ligament stem cells. Animal experiments confirmed that ephrinB2-modified canine periodontal ligament stem cells + PuraMatrix resulted in greater trabecular bone volume per tissue volume and trabecular thickness compared with other groups. Our study demonstrated that ephrinB2 promoted osteogenic differentiation of periodontal ligament stem cells and alveolar bone repair in beagles, highlighting its therapeutic potential for the treatment of alveolar bone damage.

Shear stress promotes differentiation of stem cells from human exfoliated deciduous teeth into endothelial cells via the downstream pathway of VEGF-Notch signaling.

Effects of shear stress on endotheliaxl differentiation of stem cells from human exfoliated deciduous teeth (SHEDs) were investigated. SHEDs were treated with shear stress, then reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to analyse the mRNA expression of arterial markers and western blot analysis was performed to analyse protein expression of angiogenic markers. Additionally, in vitro matrigel angiogenesis assay was performed to evaluate vascular-like structure formation. The secreted protein expression levels of the vascular endothelial growth factor (VEGF) of SHEDs after shear stress was also quantified using corresponding ELISA kits. Untreated SHEDs seeded on Matrigel cannot form vessel-like structures at any time points, whereas groups treated with shear stress formed a few vessel-like structures at 4, 8 and 12 h. When SHEDs were treated with EphrinB2-siRNA for 24, the capability of vessel-like structure formation was suppressed. After being treated with shear stress, the expression of VEGF, VEGFR2, DLL4, Notch1, EphrinB2, Hey1 and Hey2 (arterial markers) gene expression was significantly upregulated, moreover, the protein levels of VEGFR2, EphrinB2, CD31, Notch1, DLL4, Hey1, and Hey2 were also significantly up-regulated. Both the mRNA and protein expression levels of EphB4 (venous marker) were downregulated. The average VEGF protein concentration in supernatants secreted by shear stress treated SHEDs groups increased significantly. In conclusion, shear stress was able to induce arterial endothelial differentiation of stem cells from human exfoliated deciduous teeth, and VEGF-DLL4/Notch­EphrinB2 signaling was involved in this process.

[Effect of transfected EphB4 on osteogenic differentiation in stem cells from human exfoliated deciduous teeth].

PURPOSE: To evaluate the effect of stem cells from human exfoliated deciduous teeth (SHED) transfected with EphB4 gene in regulating osteogenic differentiation. METHODS: Human dental pulp tissue were harvested from extracted deciduous teeth and digested by collagenase and dispase. The SHEDs were transfected with transgenic (hEphB4-GFP) vector or empty vector (GFP-vector). Real time-polymerase chain reaction（real time-PCR） analysis and Western blot were used to detect the expression of EphB4 in SHEDs after transfection. EphB4-SHEDs and GFP-SHEDs were subjected to osteogenic induction and assessed by alkaline phosphatase(ALP) assay and Alizarin-red S staining. SPSS 16.0 software package was used for statistical analysis. RESULTS: Real time-PCR revealed that the expression of EphB4 was significantly enhanced in EphB4-SHEDs compared to GFP-SHEDs (P<0.05). The expression of EphB4 protein was significantly higher (P<0.05) in EphB4-SHEDs compared to GFP-SHEDs. ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EphB4-SHEDs. CONCLUSIONS: The results indicate that transgenic expression of EphB4 in SHEDs could promote osteogenic differentiation.

[The mechanism of corticotomy accelerating orthodontic tooth movement in SD rats].

PURPOSE: To study the influences of corticotomy on orthodontic tooth movement (OTM) and the underlying mechanism in rats. METHODS: Forty-eight female Sprague-Dawley rats were randomly assigned to corticotomy group (Cort, 24 rats) or sham-corticotomy group (Sham, 24 rats). All rats were subjected OTM after corticotomy or sham surgery. 6 rats of each group were sacrificed at 0, 1, 3 and 7 day of OTM. OTM were measured with an electronic digital caliper. Osteoclasts were counted in pressure side with TRAP. RANKL were measure by IHC at pressure side. The data were analyzed with SPSS 16.0 software package. RESULTS: OTM at the 1 and 7 day in Cort group increased compared with sham group. Number of TRAP positive osteoclasts in pressure side increased in Cort group at the 3 and 7 day. Expression of RANKL in pressure side also increased in Cort group at the 3 and 7 day. CONCLUSIONS: Corticotomy accelerates OTM in rats and it may result from promoted bone resorption via increased RANKL expression in periodontal tissue.

Decellularized extracellular matrix of human umbilical vein endothelial cells promotes endothelial differentiation of stem cells from exfoliated deciduous teeth.

Dental stem cells can serve as a potential source of functional endothelial cells for tissue engineering applications, but the endothelial-lineage differentiation efficiency is rather low even with growth factors and mechanical stimuli, which greatly limits their clinical applications. This is partly due to the deficiency of standard two-dimensional (2-D) culture systems, which is unable to recapitulate the three-dimensional (3-D) in vivo milieu that is rich in extracellular matrix. Hence, we extracted decellularized extracellular matrix from human umbilical vein endothelial cells (HUVECs-DECM) to provide a bioactive substratum conducive to the endothelial differentiation of dental stem cells. Compared to cells plated on tissue culture polystyrene (TCP), stem cells from exfoliated deciduous teeth (SHED) cultured on the HUVECs-DECM demonstrated more regular arrangement and elongated morphology. HUVECs-DECM significantly enhanced the rapid adhesion and proliferation rates of SHED, as demonstrated by WST-8 assay and immunocytochemistry indicating higher expression levels of vinculin by newly adherent SHED on HUVECs-DECM versus TCP. In addition, there was twofold to fivefold higher mRNA expression levels of endothelial-specific markers CD31 and VEGFR-2 in SHED after seven days of culture on DECM versus TCP. Functional testing with in vitro matrigel angiogenesis assay identified more capillary-like structure formation with significantly higher tubule length in SHED induced by DECM versus TCP. Hence, the results of this study provide a better understanding of the unique characteristics of cell-specific ECM and demonstrated the potential use of HUVECs-DECM as a culture substratum conducive for stimulating the endothelial differentiation of SHED for therapeutic angiogenic applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 1083-1093, 2017.

Marginal bone loss around dental implants with and without microthreads in the neck: A systematic review and meta-analysis.

STATEMENT OF PROBLEM: Whether microthreads in the crestal portion can reduce the amount of marginal bone loss (MBL) around implants has not yet been determined. PURPOSE: The purpose of this systematic review was to investigate the marginal bone loss around dental implants with and without microthreads in the neck. MATERIAL AND METHODS: This review was based on the PRISMA guidelines. An electronic search with no restrictions on language was performed from inception to August 19, 2015, in PubMed, Cochrane Central Register of Controlled Trials, EMBASE, Web of Sciences, and AMED (Ovid) databases. A manual search was also performed. Randomized clinical trials (RCTs) that compared the MBL between implants with and without microthreads in the neck were included. Qualitative synthesis and meta-analysis were performed. MBL was measured by using the mean difference (MD). Review Manager v5.3 software was used for meta-analysis (α=.05). RESULTS: Five articles were included in the qualitative synthesis, and 3 articles were included in the meta-analysis. Four studies found that a microthread design can significantly reduce MBL under functional loading, whereas 1 study found no significant difference. The homogeneity test of meta-analysis confirmed acceptable heterogeneity among the 3 studies (I2=0.49). A random-effects model was used. The result shows that MBL around implants with microthread design can be reduced significantly (P=.030; MD: -0.09; CI: -0.18 to -0.01). CONCLUSIONS: Meta-analysis showed that microthread design in the implant neck can reduce the amount of MBL; however, RCTs included in the review were few and the difference was small. In clinical practice, an implant with a roughened surface and microthreaded neck could be selected to maintain bone level.

EphrinB2 Stabilizes Vascularlike Structures Generated by Endothelial Cells and Stem Cells from Apical Papilla.

INTRODUCTION: This study aimed to investigate the roles of ephrinB2 in stabilizing vascularlike structures generated by stem cells from apical papilla (SCAPs) and human umbilical vein endothelial cells (HUVECs). METHODS: HUVECs were seeded alone or with SCAPs concurrently or 12 hours later. Angiogenesis and ephrinB2 phosphorylation were assayed at different time points. Additionally, ephrinB2 expression in SCAPs and HUVECs was silenced with small interfering RNA, and vascularlike structure formation within coculture was assessed; 1 × 10(5) HUVECs were seeded in transwell inserts, and 6 × 10(5) SCAPs were plated in lower wells with or without ephrinB2-Fc. Migratory cells were stained and counted. Delayed addition of ephrinB2-Fc to the coculture of HUVECs and SCAPs was performed to evaluate the role of ephrinB2 on the stabilization of vascularlike structures. RESULTS: Concurrent coculture of SCAPs and HUVECs yielded significantly longer tubule lengths at 4, 8, and 12 hours (P < .05). Delayed addition of SCAPs to coculture with HUVECs resulted in vascularlike structures persisting longer than the HUVEC monoculture. Western blot confirmed that ephrinB2 phosphorylation was initiated at 0.5 hours of coculture and peaked at 1 hour. Silencing ephrinB2 expression in SCAPs and HUVECs resulted in the absence of vascularlike structures. Enhanced migration of HUVECs by SCAPs could be inhibited by ephrinB2-Fc. When ephrinB2-Fc was added at 3 hours of coculture, the vascularlike structures were stabilized for more than 12 hours as compared with 9 hours in the control group. CONCLUSIONS: EphrinB2 plays an important role in the stabilization of vascularlike structures generated by HUVECs and SCAPs.

Effects of decellularized matrices derived from periodontal ligament stem cells and SHED on the adhesion, proliferation and osteogenic differentiation of human dental pulp stem cells in vitro.

A major bottleneck to the therapeutic applications of dental pulp stem cells (DPSC) are their limited proliferative capacity ex vivo and tendency to undergo senescence. This may be partly due to the sub-optimal in vitro culture milieu, which could be improved by an appropriate extracellular matrix substratum. This study therefore examined decellularized matrix (DECM) from stem cells derived from human exfoliated deciduous teeth (SHED) and periodontal ligament stem cells (PDLSC), as potential substrata for DPSC culture. Both SHED-DECM and PDLSC-DECM promoted rapid adhesion and spreading of newly-seeded DPSC compared to bare polystyrene (TCPS), with vinculin immunocytochemistry showing expression of more focal adhesions by newly-adherent DPSC cultured on DECM versus TCPS. Culture of DPSC on SHED-DECM and PDLSC-DECM yielded higher proliferation of cell numbers compared to TCPS. The qRT-PCR data showed significantly higher expression of nestin by DPSC cultured on DECM versus the TCPS control. Osteogenic differentiation of DPSC was enhanced by culturing on PDLSC-DECM and SHED-DECM versus TCPS, as demonstrated by alizarin red S staining for mineralized calcium deposition, alkaline phosphatase assay and qRT-PCR analysis of key osteogenic marker expression. Hence, both SHED-DECM and PDLSC-DECM could enhance the ex vivo culture of DPSC under both non-inducing and osteogenic-inducing conditions.