C Zhang

Human umbilical vein endothelial cells synergize osteo/odontogenic differentiation of periodontal ligament stem cells in 3D cell sheets

BACKGROUND AND OBJECTIVE To investigate the expression of osteo/odontogenic differentiation markers and vascular network formation in a 3D cell sheet with varying cell ratios of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs). MATERIAL AND METHODS Human PDLSCs were isolated and characterized by flow cytometry, and co-cultured with HUVECs for the construction of cell sheets. Both types of cells were seeded on temperature-responsive culture dishes with PDLSCs alone, HUVECs alone and various ratios of the latter cells (1 : 1, 2 : 1, 5 : 1 and 1 : 5) to obtain confluent cell sheets. The expressions of osteo/odontogenic pathway markers, including alkaline phosphatase (ALP), bone sialoprotein (BSP) and runt-related transcription factor 2 (RUNX2), were analyzed at 3 and 7 d using RT-PCR. Further ALP protein quantification was performed at 7 and 14 d using ALP assay. The calcium nodule formation was assessed qualitatively and quantitatively by alizarin red assay. Histological evaluations of three cell sheet constructs treated with different combinations (PDLSC-PDLSC-PDLSC/PDLSC-HUVEC-PDLSC/co-culture-co-culture-co-culture) were performed with hematoxylin and eosin and immunofluorescence staining. Statistical analysis was performed using t-test (p < 0.05). RESULTS Significantly higher ALP gene expression was observed at 3 d in 1 : 1 (PDLSC-HUVEC) (2.52 ± 0.67) and 5 : 1 (4.05 ± 1.07) co-culture groups compared with other groups (p < 0.05); this was consistent with ALP protein quantification. However, the expression of BSP and RUNX2 genes was higher at 7 d compared to 3 d. Significant calcium mineralization was detected as quantified by alizarin red assay at 14 d in 1 : 1 (1323.55 ± 6.54 μm) and 5 : 1 (994.67 ± 4.15 μm) co-cultures as compared with monoculture cell sheets (p < 0.05). Hematoxylin and eosin and CD31 immunostaining clearly exemplified the development of a layered cell sheet structure with endothelial cell islands within the constructed PDLSC-HUVEC-PDLSC and co-culture groups. Furthermore, HUVECs invaded the layered cell sheet, suggestive of rudimentary vascular network initiation. CONCLUSION This study suggests that the PDLSC-HUVEC co-culture, cell sheet, model exhibits significantly high levels of osteo/odontogenic markers with signs of initial vascular formation. This novel 3D cell sheet-based approach may be potentially beneficial for periodontal regenerative therapy.

In vivo periodontal tissue regeneration by periodontal ligament stem cells and endothelial cells in three-dimensional cell sheet constructs

BACKGROUND AND OBJECTIVE Chronic periodontitis causes damage to tooth-supporting tissues, resulting in tooth loss in adults. Recently, cell-sheet-based approaches have been studied to overcome the limitations of conventional cytotherapeutic procedures for periodontal regeneration. The purpose of the present study was to investigate the regenerative potential of periodontal ligament stem cells (PDLSCs) and human umbilical vein endothelial cells (HUVECs) in three-dimensional (3D) cell sheet constructs for periodontal regeneration in vivo. MATERIAL AND METHODS PDLSCs, HUVECs or co-cultures of both cells were seeded onto temperature-responsive culture dishes, and intact cell sheets were fabricated. Cell sheets were wrapped around the prepared human roots in three different combinations and implanted subcutaneously into immunodeficient mice. RESULTS Histological evaluation revealed that after 2, 4 and 8 wk of implantation, periodontal ligament-like tissue arrangements were observed around the implanted roots in experimental groups compared with controls. Vascular lumens were also observed in periodontal compartments of HUVEC-containing groups. Periodontal ligament regeneration, cementogenesis and osteogenesis were evident in the experimental groups at both weeks 4 and 8, as shown by immunostaining for periostin and bone sialoprotein. Human cells in the transplanted cell sheets were stained by immunohistochemistry for the presence of human mitochondria. CONCLUSIONS The 3D cell sheet-based approach may be potentially beneficial and is thus encouraged for future regenerative periodontal therapy.

The effectiveness of low-level laser therapy as an adjunct to non-surgical periodontal treatment: a meta-analysis.

Background and Objectives Although low‐level laser therapy (LLLT) has been demonstrated to have a biomodulatory effect on periodontal tissue, no systematic review has exclusively addressed its effectiveness as an adjunct to non‐surgical periodontal treatment. This study aimed to evaluate whether an additional benefit exists for the application of LLLT compared with scaling and root planing (SRP) alone. Material and Methods An extensive search was conducted in the Cochrane Library (Issue 8, 2015), PubMed (1997) and EMBASE (1947) before August 2015 for randomized controlled trials (RCTs). The bias risk was assessed with the Cochrane tool for risk of bias evaluation. A meta‐analysis was performed using REVMAN 5.3. Results After independent screening of 354 initial records, eight publications (seven RCTs) were included. However, six were rated as ‘having a high risk of bias’ as a result of major methodological weakness in ‘allocation concealment’ and ‘blinding of key personnel’. Meta‐analysis showed that LLLT‐mediated SRP demonstrated significant short‐term benefits over SRP monotherapy in the improvement of the probing pocket depth (p = 0.0009 at 1 mo; p = 0.03 at 2 mo) and the level of interleukin‐1β in the gingival crevicular fluid (p = 0.01 at 1 mo). Nevertheless, LLLT failed to show significant additional intermediate‐term (3 and 6 mo) effects in terms of clinical parameters and alveolar bone density. Conclusion These findings indicated that LLLT showed only short‐term additional benefits after conventional SRP. Its long‐term effects remain unclear due to substantial methodological weaknesses and an insufficient number of current studies. Future RCTs with better designs and longer follow‐up periods are required to assess the effectiveness of LLLT as an adjunctive treatment strategy in patients with periodontal disease.

Expression profile of macrophage migration-inhibitory factor in human gingiva and reconstituted human gingival epithelia stimulated by Porphyromonas gingivalis lipopolysaccharide

BACKGROUND AND OBJECTIVE Macrophage migration-inhibitory factor (MIF) plays crucial roles in the recruitment and activation of macrophages as well as in helping to kill bacteria. This study investigated the expression profile of MIF in human gingiva under different periodontal conditions and its expression patterns induced by Porphyromonas gingivalis lipopolysaccharide (LPS) in gingival epithelia. MATERIAL AND METHODS Gingival tissue samples were collected from deep pockets and clinically healthy sites of 22 nonsmoking subjects with chronic periodontitis. The expression of MIF mRNA and protein was evaluated using real-time PCR and immunohistochemistry, respectively. The in vitro study analyzed the effects of P. gingivalis LPS on the expression of MIF in a reconstituted human gingival epithelia (RHGE) model. RESULTS In gingival epithelia, MIF protein was diffusely expressed from the basal layer to the granular and spinous layers; whereas, in the underlying connective tissues, MIF was observed around the dilated blood vessels in the deep-pocket tissues. A significantly lower level of expression of MIF mRNA and an increased level of expression of MIF protein were found in deep-pocket tissues compared with clinically healthy tissues. Expression of MIF mRNA in the RHGE model was significantly down-regulated by P. gingivalis LPS. CONCLUSION The present study suggests that MIF expression may be related to periodontal conditions and that its expression profile could be modulated by P. gingivalis LPS. MIF may play a role in periodontal pathogenesis.

Transgenic expression of ephrinB2 in periodontal ligament stem cells (PDLSCs) modulates osteogenic differentiation via signaling crosstalk between ephrinB2 and EphB4 in PDLSCs and between PDLSCs and pre‐osteoblasts within co‐culture

BACKGROUND AND OBJECTIVE The goal of periodontal therapy is to regenerate/reconstruct the damaged supporting tissues of diseased teeth and to facilitate recovery of their physiological functions. Combination of stem cell transplantation and gene therapy offers a viable method for accelerating periodontal repair and regeneration. In this study, the role of the ephrinB2/EphB4 signaling pathway in regulating osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and crosstalk between PDLSCs and pre-osteoblasts within co-culture was investigated through ephrinB2 transgenic expression in PDLSCs. MATERIAL AND METHODS PDLSCs isolated from premolar teeth of teenage patients undergoing orthodontic treatment were transfected with transgenic (hEfnB2-GFP-Bsd) vector or empty vector (GFP-Bsd). Vector-PDLSCs, EfnB2-PDLSCs, MC3T3-E1 and co-cultures of vector-PDLSCs with MC3T3-E1, and EfnB2-PDLSCs with MC3T3-E1 were subjected to osteogenic induction. The osteogenic differentiation of EfnB2-PDLSCs, vector-PDLSCs and co-cultures were assessed by reverse transcription-polymerase chain reaction, alkaline phosphatase (ALP) assay and Alizarin-red S staining. Protein expression levels of ephrinB2, EphB4, phosphorylated ephrinB2 and EphB4 were analyzed by western blot, immunoprecipitation and co-immunoprecipitation assays. RESULTS ALP assay and Alizarin-red S staining demonstrated higher ALP activity and increased mineralization with EfnB2-PDLSCs vs. vector-PDLSCs and with co-culture of EfnB2-PDLSCs and MC3T3-E1 vs. vector-PDLSCs and MC3T3-E1. Reverse transcription-polymerase chain reaction revealed that the expression of human odonto/osteogenic markers were significantly enhanced in EfnB2-PDLSCs compared to vector-PDLSCs, and that the expression of mouse odonto/osteogenic markers were significantly higher in co-culture of EfnB2-PDLSCs with MC3T3-E1 vs. vector-PDLSCs with MC3T3-E1. The EphB4 receptor was activated through phosphorylation during osteogenic differentiation. CONCLUSION Our data indicate that transgenic expression of ephrinB2 in PDLSCs could promote osteogenic differentiation via stimulation of the phosphorylation of ephrinB2 and EphB4, which regulates cell communication between PDLSCs and between PDLSCs and pre-osteoblasts within co-culture.

The spherical nanoparticle-encapsulated chlorhexidine enhances anti-biofilm efficiency through an effective releasing mode and close microbial interactions

We reported two forms (sphere and wire) of newly fabricated chlorhexidine (CHX)-loaded mesoporous silica nanoparticles (MSNs), and investigated their releasing capacities and anti-biofilm efficiencies. The interactions of the blank MSNs with planktonic oral microorganisms were assessed by field emission scanning electron microscopy. The anti-biofilm effects of the two forms of nanoparticle-encapsulated CHX were examined by 2,3-bis (2-methoxy- 4-nitro-5-sulfo-phenyl)-2H-tetrazolium-5-carboxanilide. The profiles of biofilm penetration were analyzed by fluorescent-labeled MSNs using confocal microscopy and ImageJ. The spherical MSNs with an average diameter of 265 nm exhibited a larger surface area and faster CHX-releasing rate than the MSN wires. The field emission scanning electron microscopy images showed that both shaped MSNs enabled to attach and further fuse with the surfaces of testing microbes. Meanwhile, the nanoparticle-encapsulated CHX could enhance the anti-biofilm efficiency with reference to its free form. Notably, the spherical nanoparticle-encapsulated CHX presented with a greater anti-biofilm capacity than the wire nanoparticle-encapsulated CHX, partly due to their difference in physical property. Furthermore, the relatively even distribution and homogeneous dispersion of spherical MSNs observed in confocal images may account for the enhanced penetration of spherical nanoparticle-encapsulated CHX into the microbial biofilms and resultant anti-biofilm effects. These findings reveal that the spherical nanoparticle-encapsulated CHX could preferably enhance its anti-biofilm efficiency through an effective releasing mode and close interactions with microbes.

Periodontal therapy decreases serum levels of adipocyte fatty acid-binding protein in systemically healthy subjects: a pilot clinical trial

BACKGROUND AND OBJECTIVE Adipocyte fatty acid-binding protein (A-FABP) is expressed in adipocytes, macrophages and microvascular endothelial cells, and it plays a central role in inflammation, atherosclerosis and metabolic responses. This pilot study investigated the effect of nonsurgical periodontal therapy on the serum levels of A-FABP in subjects with chronic periodontitis. MATERIAL AND METHODS A pilot clinical trial was conducted in 24 otherwise healthy Chinese subjects with moderate to severe chronic periodontitis. The treatment group (n = 12) received nonsurgical periodontal therapy immediately, whereas in the control group (n = 12) the treatment was delayed for 3 months. The serum levels of A-FABP were measured by ELISAs. Other inflammatory and endothelial biomarkers and periodontal conditions were evaluated at baseline and at the 3-month follow-up appointment. RESULTS A-FABP levels decreased significantly in the treatment group compared with the control group (treatment effect: -1.7 ng/mL; 95% confidence interval: -2.8 to -0.6; p = 0.003). The treatment also significantly improved periodontal conditions but had no significant effect on other biomarkers. In the multivariable regression model, the change in the percentage of sites with detectable plaque was significantly associated with the change in the level of A-FABP (beta: 0.04, 95% confidence interval: 0.01-0.06, p = 0.004). CONCLUSION Within the limitations of this pilot study, the current findings suggest that treatment of periodontitis may significantly decrease the serum levels of A-FABP. Further longitudinal study with a large sample size is warranted to confirm this finding and elaborate the relevant clinical implications.

Porphyromonas gingivalis lipopolysaccharide regulates ephrin/Eph signalling in human periodontal ligament fibroblasts

Objective EphrinA2‐EphA2 and ephrinB2‐EphB4 critically engage in bidirectional signalling to modulate alveolar bone remodelling. The present study aimed to investigate the effects of lipopolysaccharides (LPS) derived from Porphyromonas gingivalis on ephrin/Eph signalling in periodontal ligament fibroblasts (PDLFs). Material and Methods The primary cultured PDLFs were incubated in the absence (as a control) or presence of P. gingivalis LPS at 0.001‐10 μg/mL for 24 hours. The PDLFs were then stimulated with P. gingivalis LPS at the optimal concentration (0.1 μg/mL) for different periods (6‐48 hours). The expression of ephrinA2, ephrinB2, EphA2 and EphB4 was assessed by quantitative reverse‐transcription real‐time polymerase chain reaction and western blotting. The osteoblastic markers alkaline phosphatase, osteocalcin and Runt‐related transcription factor 2 (Runx2), and the osteoclastogenesis‐related factors receptor activator of nuclear factor kappa‐B ligand (RANKL) and osteoprotegerin were also evaluated. Results The ephrinA2 and EphA2 expression was upregulated and EphB4 expression was downregulated by stimulation of P. gingivalis LPS. EphrinA2 mRNA expression in the PDLFs was significantly upregulated from 12 to 48 hours (P<.05), whereas EphA2 exhibited no change for the first 24 hours, after which there was a significant increase at 48 hours (P<.05). EphB4 exhibited lower mRNA expression at 12 and 24 hours than did the control (P<.05), but the change was insignificant at 48 hours. In contrast, the expression of ephrinB2 remained unchanged. The expressions of ephrinA2, EphA2, ephrinB2 and EphB4 at the protein level showed a similar pattern to that at the mRNA level. The expression of Runx2 and osteocalcin significantly decreased, whereas that of RANKL/osteoprotegerin increased. Conclusion The present study suggest that P. gingivalis LPS would contribute to a dysregulation of bone remodelling, whereby ephrinA2/EphA2 expression is stimulated and EphB4 expression is inhibited.