[Show abstract][Hide abstract] ABSTRACT: To study the immune-inflammatory response and signaling mechanism of macrophages to purified Enterococcus faecalis (E. faecalis) lipoteichoic acid (LTA), intact LTA was obtained from an E. faecalis clinical strain P25RC using the butanol method and hydrophobic interaction chromatography purification. The fractions containing LTA were determined using phosphate detection. Contaminations with lipopolysaccharide and proteins were excluded using the Limulus amoebocyte lysate assay and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively. LTA was analyzed using nuclear magnetic resonance. Prior to LTA stimulation assays, THP-1 monocytes were pretreated with phorbol 12-myristate 13-acetate to differentiate into macrophages. Macrophages were treated with LTA in concentration gradients and cells without LTA treatment as the control. Gene expression of TLR2, CD14 and MyD88 were evaluated by quantitative polymerase chain reaction. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-10 were quantified using ELISA. The activated and total nuclear factor-κB (NF-κB) p65 and three mitogen-activated protein kinases (p38, ERK1/2 and JNK) were assessed using western blot analysis. E. faecalis LTA induced the gene expression of TLR2 and MyD88 whilst it downregulated CD14, suggesting a TLR2-dependent and CD14-independent immune-inflammatory activity. LTA stimulated the expression of pro-inflammatory cytokine TNF-α (P<0.05), but not the anti-inflammatory cytokine IL-10. In conclusion, E. faecalis LTA stimulated the expression of TNF-α in macrophages possibly through the NF-κB and p38 pathways.
[Show abstract][Hide abstract] ABSTRACT: Candida infection has emerged as a critical health care burden worldwide, owing to the formation of robust biofilms against common
antifungals. Recent evidence shows that multidrug-tolerant persisters critically account for biofilm recalcitrance, but their
underlying biological mechanisms are poorly understood. Here, we first investigated the phenotypic characteristics of Candida biofilm persisters under consecutive harsh treatments of amphotericin B. The prolonged treatments effectively killed the
majority of the cells of biofilms derived from representative strains of Candida albicans, Candida glabrata, and Candida tropicalis but failed to eradicate a small fraction of persisters. Next, we explored the tolerance mechanisms of the persisters through
an investigation of the proteomic profiles of C. albicans biofilm persister fractions by liquid chromatography-tandem mass spectrometry. The C. albicans biofilm persisters displayed a specific proteomic signature, with an array of 205 differentially expressed proteins. The
crucial enzymes involved in glycolysis, the tricarboxylic acid cycle, and protein synthesis were markedly downregulated, indicating
that major metabolic activities are subdued in the persisters. It is noteworthy that certain metabolic pathways, such as the
glyoxylate cycle, were able to be activated with significantly increased levels of isocitrate lyase and malate synthase. Moreover,
a number of important proteins responsible for Candida growth, virulence, and the stress response were greatly upregulated. Interestingly, the persisters were tolerant to oxidative
stress, despite highly induced intracellular superoxide. The current findings suggest that delicate metabolic control and
a coordinated stress response may play a crucial role in mediating the survival and antifungal tolerance of Candida biofilm persisters.
No preview · Article · Jul 2015 · Antimicrobial Agents and Chemotherapy
[Show abstract][Hide abstract] ABSTRACT: Introduction:
Scaffolds often fail to mimic essential functions of the physiologic extracellular matrix (ECM) that regulates cell-cell communication in tissue microenvironments. The development of scaffold-free microtissues containing stem cell-derived ECM may serve as a successful alternative to the use of artificial scaffolds. The current study aimed to fabricate 3-dimensional microtissue spheroids of dental pulp cells (DPCs) prevascularized by human umbilical vein endothelial cells (HUVECs) and to characterize these scaffold-free spheroids for the in vitro formation of pulplike tissue constructs.
Three-dimensional microtissue spheroids of DPC alone and DPC-HUVEC co-cultures were fabricated using agarose micro-molds. Cellular organization within the spheroids and cell viability (live/dead assay) were assessed at days 1, 7, and 14. Microtissue spheroids were allowed to self-assemble into macrotissues, induced for odontogenic differentiation (21 days), and examined for expression levels of osteo/odontogenic markers: alkaline phosphatase, bone sialoprotein and RUNX2 (Real-time PCR), mineralization (von-Kossa), and prevascularisation (immunohistochemistry for CD31).
The DPC microtissue microenvironment supported HUVEC survival and capillary network formation in the absence of a scaffolding material and external angiogenic stimulation. Immunohistochemical staining for CD31 showed the capillary network formed by HUVECs did sustain-for a prolonged period-even after the microtissues transformed into a macrotissue. Induced, prevascularized macrotissues showed enhanced differentiation capacity compared with DPC alone macrotissues, as shown by higher osteo/odontogenic gene expression levels and mineralization.
These findings provide insight into the complex intercellular cross talk occurring between DPCs and HUVECs in the context of angiogenesis and pulp regeneration and highlight the significance of developing a favorable 3-dimensional microenvironment that can, in turn, contribute toward successful pulp regeneration strategies.
Full-text · Article · Feb 2015 · Journal of endodontics
[Show abstract][Hide abstract] ABSTRACT: The success of bioengineered dental pulp depends on two principles, (1) whether the transplanted tissue can develop its own vascular endothelial tubule network and (2) whether the host vasculature can be induced to penetrate the bioengineered pulp replacement and conjoin. Major inductive molecules that participate in laying down blood vessels include vascular endothelial growth factor (VEGF), ephrinB2, and hypoxia-inducible factor 1α (HIF-1α). Being able to modulate the genes encoding these angiogenic molecules is a therapeutic target in pulp regeneration for endogenous blood vessel formation, prevention of graft rejection, and exclusion of infection. Once implanted inside the root canal, bioengineered pulp is subjected to severe hypoxia that causes tissue degeneration. However, short-term hypoxia is known to stimulate angiogenesis. Thus, it may be feasible to prime dental cells for angiogenic activity prior to implantation. Stem cells from apical papilla (SCAP) are arguably one of the most potent and versatile dental stem cell populations for bioengineering pulp in vitro. Our study aimed to investigate whether coculture of SCAP and human umbilical vein endothelial cells (HUVEC) under hypoxia promotes formation of endothelial tubules. In addition, we clarified the interplay between the genes that orchestrate these important angiogenic molecules in SCAP under hypoxic conditions. We found that SCAP cocultured with HUVEC at a 1:5 ratio increased the number of endothelial tubules, tubule lengths, and branching points. Fluorescence staining showed that HUVEC formed the trunk of tubular structures, while SCAP located adjacent to the endothelial cell line, resembling the pericyte location. When we used CoCl2 (0.5 mM) to induce hypoxic environment, the expression of proteins, HIF-1α, and VEGF; and transcript of ephrinB2 in SCAP was upregulated. However, minimal VEGF levels in supernatants of HUVEC and coculture petri dishes were detected, suggesting that VEGF secreted by SCAP might be used by HUVEC to accelerate the formation of vessel-like structures. Taken together, we revealed that artificial hypoxia stimulates angiogenic responses in SCAP for possible use in engineering dental pulp replacements. Our results may help to delineate the optimal therapeutic target to promote angiogenesis so that future bioengineered pulp replacements integrate faster and permanently within the host.
Full-text · Article · Nov 2014 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Securing an adequate blood supply for the survival of cell transplants is critical for a successful outcome in tissue engineering. Interactions between endothelial and progenitor/stem cells are important for the vascularization of regenerating tissue. Recently, self-assembling peptide nanofibers were described as a promising environment for pulp regeneration due to their synthetic nature and controlled physicochemical properties. In the present study, the peptide hydrogel PuraMatrixTM was used as a scaffold system to investigate the role of dental pulp stem cells (DPSCs) in triggering angiogenesis and the potential for regenerating vascularized pulp in vivo. Human umbilical vein endothelial cells (HUVECs), DPSCs, or co-cultures of both cell types were encapsulated in three-dimensional PuraMatrixTM. The peptide nanofiber microenvironment supported cell survival, cell migration, and capillary network formation in the absence of exogenous growth factors. DPSCs increased early vascular network formation by facilitating the migration of HUVECs and by increasing vascular endothelial growth factor (VEGF) expression. Both the DPSC-monoculture and co-culture groups exhibited vascularized pulp-like tissue with patches of osteodentin after transplantation in mice. The co-cultured groups exhibited more extracellular matrix, vascularization, and mineralization than the DPSC-monocultures in vivo. Whereas the DPSCs play a critical role in initial angiogenesis, coordinated efforts by the HUVECs and DPSCs are required to achieve a balance between extracellular matrix deposition and mineralization. The findings of this study also highlighted the importance of a microenvironment that supports cell-cell interactions and cell migration, which contribute to successful dental pulp regeneration.
No preview · Article · Sep 2014 · Tissue Engineering Part A
[Show abstract][Hide abstract] ABSTRACT: Creating an optimal microenvironment that mimics the extracellular matrix (ECM) of natural pulp and securing an adequate blood supply for the survival of cell transplants are major hurdles that need to be overcome in dental pulp regeneration. However, many currently available scaffolds fail to mimic essential functions of natural ECM. The present study investigated a novel approach involving the use of scaffold-free microtissue spheroids of dental pulp stem cells (DPSCs) prevascularized by human umbilical vein endothelial cells (HUVECs) in pulp regeneration. In vitro-fabricated microtissue spheroids were inserted into the canal space of tooth-root slices and were implanted subcutaneously into immunodeficient mice. Histological examination revealed that, after four-week implantation, tooth-root slices containing microtissue spheroids resulted in well-vascularized and cellular pulp-like tissues, compared with empty tooth-root slices, which were filled with only subcutaneous fat tissue. Immunohistochemical staining indicated that the tissue found in the tooth-root slices was of human origin, as characterized by the expression of human mitochondria, and contained odontoblast-like cells organized along the dentin, as assessed by immunostaining for nestin and dentin sialoprotein (DSP). Vascular structures formed by HUVECs in vitro were successfully anastomosed with the host vasculature upon transplantation in vivo, as shown by immunostaining for human CD31. Collectively, these findings demonstrate that prevascularized, scaffold-free, microtissue spheroids can successfully regenerate vascular dental pulp-like tissue and also highlight the significance of the microtissue microenvironment as an optimal environment for successful pulp-regeneration strategies.
Full-text · Article · Sep 2014 · Journal of Dental Research
[Show abstract][Hide abstract] ABSTRACT: Background
Chlorhexidine (CHX) is a widely used antimicrobial agent in dentistry. Herein, we report the synthesis of a novel mesoporous silica nanoparticle-encapsulated pure CHX (Nano-CHX), and its mechanical profile and antimicrobial properties against oral biofilms.
The release of CHX from the Nano-CHX was characterized by UV/visible absorption spectroscopy. The antimicrobial properties of Nano-CHX were evaluated in both planktonic and biofilm modes of representative oral pathogenic bacteria. The Nano-CHX demonstrated potent antibacterial effects on planktonic bacteria and mono-species biofilms at the concentrations of 50–200 µg/mL against Streptococcus mutans, Streptococcus sobrinus, Fusobacterium nucleatum, Aggregatibacter actinomycetemcomitans and Enterococccus faecalis. Moreover, Nano-CHX effectively suppressed multi-species biofilms such as S. mutans, F. nucleatum, A. actinomycetemcomitans and Porphyromonas gingivalis up to 72 h.
This pioneering study demonstrates the potent antibacterial effects of the Nano-CHX on oral biofilms, and it may be developed as a novel and promising anti-biofilm agent for clinical use.
[Show abstract][Hide abstract] ABSTRACT: Objectives: To co-culture dental pulp stem cells (DPSCs) and human umbilical vein endothelial cells (HUVECs) in an injectable, peptide hydrogel scaffold: PuraMatrix while fabricating a vascular network in-vitro and to utilize this construct in pulp regeneration in-vivo.
Methods: DPSCs and HUVECs were encapsulated in PuraMatrix (BD-Biosciences) as mono-cultures or co-cultures at different ratios (3:1, 1:1 1:3). Viability, morphology and three-dimensional organization of cells within PuraMatrix were assessed over 2-weeks under confocal microscopy. Cell-PHS constructs were induced for odonto/osteogenic differentiation (up to 21-days); and examined for alkaline phosphatase (ALP) activity and mineralization (von-Kossa staining). Cell-encapsulated PuraMatrix-constructs were injected into the canal space of full-length tooth-roots and implanted into the subcutaneous space of 6-8-week-old female severe combined immunodeficient mice. Two-to-four weeks after transplantation, mice were euthanized and tooth-roots were removed for histological (Haematoxylin and eosin) and immunohistochemical (human mitochondria, CD31) analysis. Experiments were conducted in triplicate using DPSCs from three different donors and analysed statistically (ANOVA).
Results: Results showed that both DPSCs and HUVECs survived well in co-cultures compared to monocultures. HUVECs, when co-cultured with DPSCs formed a vessel-like network throughout the PuraMatrix compared to HUVEC-monocultures where not only HUVECs failed to form any vessels but also underwent apoptosis. ELISA-assay revealed that DPSCs secrete VEGF in high amounts inhibiting apoptosis and promoting vessel formation by HUVECs. Higher ALP activity and mineralization were observed in co-cultures compared to monocultures (p<0.05). Both DPSC-monoculture and co-culture groups showed vascularised pulp-like tissue with patches of osteodentin after transplantation in mice. Co-cultured groups showed higher amounts of extracellular matrix, vascularisation and mineralization compared to DPSC-monocultures in-vivo. Immunohistochemistry for human mitochondria confirmed the contribution of transplanted cells in regenerated pulp-like tissue and vasculature.
Conclusion: DPSCs and HUVECs being encapsulated within PuraMatrix demonstrate synergistic effects in odontogenic differentiation and angiogenesis and have potential for engineering vascularised pulp tissues in-vivo.
[Show abstract][Hide abstract] ABSTRACT: This present study was designed to investigate the effects of Angiotensin II on mitochondrial functions, ROS generation and c-jun N-terminal kinases (JNK) signalling pathway-mediated cell apoptosis in mouse calvaria osteoblasts.
Calvaria osteoblast were isolated and cultured. The cells were separated into two groups-control and treated groups-where the latter was stimulated with angiotensin II (Ang II). Mitochondrial reactive oxygen species (ROS) and superoxide production were measured. Intracellular ATP levels were also detected. The cell proliferation rate was determined for the two groups. Protein production such as Anti-Bax, Bcl-2, COX IV and activation of c-jun N-terminal kinases signal (JNK) pathway was measured by enzyme-linked immunosorbent assay (ELISA) methods and Western blotting in this study.
Ang II treated cells showed significantly higher levels of superoxide production compared to the control group (p<0.05). Conversely, Ang II induced inhibitory effects on mitochondrial respiratory enzyme complexes, cause membrane potential dissipation, ATP loss and promote ROS generation, cell apoptosis in cultured osteoblasts. In addition, JNK phosphorylations were involved in activating the mitochondria-dependent apoptotic pathway following Ang II stimulation, as pre-treatment of JNK-specific inhibitor SP600125 could rescue osteoblast cells from apoptosis by enhancing the anti-apoptotic protein Bcl-2 expressions, suppressing the translocation of Bax from cytosol into mitochondria, blocking cytochrome C release and caspase-3 activation.
Ang II stimulates osteoblast apoptosis via suppression of the mitochondrial respiratory enzymes, membrane potential and cellular ATP productions. Clinical application with Ang II-stimulated osteoblast could be used for modelling or bone resorption in the oral region.
No preview · Article · Mar 2014 · Archives of oral biology
[Show abstract][Hide abstract] ABSTRACT: Background
Porphyromonas gingivalis lipopolysaccharide (LPS) is a crucial virulence factor strongly associated with chronic periodontitis which is the primary cause of tooth loss in adults. It exhibits remarkable heterogeneity containing tetra-(LPS1435/1449) and penta-(LPS1690) acylated lipid A structures. Human gingival fibroblasts (HGFs) as the main resident cells of human gingiva play a key role in regulating matrix metalloproteinases (MMPs) and contribute to periodontal homeostasis. This study investigated the expression and regulation of MMPs1-3 and tissue inhibitors of MMP-1 (TIMP-1) in HGFs in response to P. gingivalis LPS1435/1449 and LPS1690 and hexa-acylated E. coli LPS as a reference. The expression of MMPs 1–3 and TIMP-1 was evaluated by real-time PCR and ELISA.
The MMP-3 mRNA and protein were highly upregulated in P. gingivalis LPS1690- and E. coli LPS-treated cells, whereas no induction was observed in P. gingivalis LPS1435/1449-treated cells. On the contrary, the expression of MMP-1 and −2 was not significantly affected by P. gingivalis LPS lipid A heterogeneity. The TIMP-1 mRNA was upregulated in P. gingivalis LPS1435/1449- and E. coli LPS-treated cells. Next, signal transduction pathways involved in P. gingivalis LPS-induced expression of MMP-3 were examined by blocking assays. Blockage of p38 MAPK and ERK significantly inhibited P. gingivalis LPS1690-induced MMP-3 expression in HGFs.
The present findings suggest that the heterogeneous lipid A structures of P. gingivalis LPS differentially modulate the expression of MMP-3 in HGFs, which may play a role in periodontal pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: P. gingivalis (Pg) LPS -induced gene expression of inflammatory mediators in HGFs. The cells were treated with PgLPS at 1 µg/mL or culture medium alone for 24 h. Total RNA was extracted and reverse transcribed into cDNA templates. The templates used in PCR array were pooled equally from triplicate samples. Representative heat maps showing the fold-changes of each gene in PgLPS1435 (A)- and PgLPS1690 (B)-treated HGFs with reference to the controls. Genes that were upregulated over 2 folds are shown in red color and those down regulated by 0.5 folds are shown in green color.
[Show abstract][Hide abstract] ABSTRACT: Confocal images of TLR2 (S1.1) and TLR4 (S1.2) expression in HGFs following LPS stimulation for 24 h. HGFs were left untreated (A) or stimulated with 1 µg/ml of P. gingivalis (Pg) LPS1435/1449 (PgLPS1435/1449) (B) PgLPS1690 (C) and E. coli LPS (D). Negative control: E. Cells were then permeabilized with 0.1% Triton X-100 and subsequently stained with primary antibodies against TLR2, TLR4 and the correspondent secondary antibody labeled Alexa fluor 488 anti-rabbit, and subsequently stained with alexa fluor 555 phalloidin for F-actin. Merged images present the combined TLR2 or TLR4, F-actin, and nuclear staining (DAPI). One representative experiment from three independent experiments is shown. Bar = 50 µm or 100 µm.
[Show abstract][Hide abstract] ABSTRACT: Differential expression profile of genes associated with TLR signal transduction in HGF. The cells were treated with P. gingivalis (Pg) LPS1435/1449 (PgLPS1435/1449) and PgLPS1690 (1 µg/mL) for 24 h. After the stimulation, mRNA was extracted from cellular fraction and reverse transcribed to cDNA. Pathway-focused PCR gene array was adopted to analyse the cDNA corresponding to 84 inflammation-associated genes quantified with qRT-PCR. Relative expression was analysed comparing the LPS treated cells with the cDNA prepared from the controls. The fold-changes in gene expression in the P. gingivalis LPS-treated cells versus control cells are listed. Genes that were upregulated over 2 folds are marked in red color and those down regulated by 0.5 folds are highlighted in blue color.
[Show abstract][Hide abstract] ABSTRACT: Kinetics of protein kinase B (PKB) or AKT phosphorylation in HGFs. The cells were stimulated with P. gingivalis (Pg) LPS1435/1449
(PgLPS1435/1449) (A), PgLPS1690 (B) and E. coli LPS (C) at 1 µg/mL for the indicated periods of time. Cell extracts were prepared and the sample aliquots containing 40 µg of protein were separated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-phopho AKT specific antibodies. Fold increase values of p-AKT optical density (arbitrary units over control after normalization to the loading control (total AKT) are shown in the graphs (D). The data shown here are from a representative experiment repeated three times with similar results. *Significant difference with a p-value <0.05 as compared with the controls without LPS treatment.
[Show abstract][Hide abstract] ABSTRACT: Genes included in the TLR signaling pathway RT-PCR array kit (SA Biosciences). A total of 84 genes related to TLR signaling family were analyzed, including adaptor and effector proteins, members of the NF-κB, JNK/p38, IRF and JAK/STAT signaling pathways as well as downstream pathway genes.
[Show abstract][Hide abstract] ABSTRACT: Nucleotide sequence of primers for real-time PCR. Quantitative real time (QRT) PCR was performed using custom-designed primers for the cell surface receptors, adaptor molecules and pro-inflammatory cytokines using purified RNA from HGFs stimulated with P. gingivalis LPS and E. coli LPS.
[Show abstract][Hide abstract] ABSTRACT: P. gingivalis (Pg) LPS1690 induced the mRNA expression of inflammatory mediators in HGFs. The cells were stimulated with PgLPS and E. coli LPS (1 µg/mL) for 24 h. The harvested RNA was subjected to real-time quantitative PCR analysis. Fold increase of genes were analyzed relative to the internal control β-Actin, including GM-CSF (A), CXCL10 (B), IL-6 (C) and IL-8 (D). Each bar represents the mean±SD of three independent experiments with three replicates. *Significant difference with a p-value <0.05 as compared with the controls without LPS treatment.
[Show abstract][Hide abstract] ABSTRACT: Porphyromonas gingivalis is a major pathogen of periodontal disease that affects a majority of adults worldwide. Increasing evidence shows that periodontal disease is linked to various systemic diseases like diabetes and cardiovascular disease, by contributing to increased systemic levels of inflammation. Lipopolysaccharides (LPS), as a key virulent attribute of P. gingivalis, possesses significant amount of lipid A heterogeneity containing tetra- (LPS1435/1449) and penta-acylated (LPS1690) structures. Hitherto, the exact molecular mechanism of P. gingivalis LPS involved in periodontal pathogenesis remains unclear, due to limited understanding of the specific receptors and signaling pathways involved in LPS-host cell interactions.
[Show abstract][Hide abstract] ABSTRACT: Commensal bacteria have been shown to be active participants in the development of the structure and function of host tissues (Gordon and Pesti, 1971; Hooper et al., 2001; Macpherson and Harris, 2004; Umesaki and Setoyama, 2000; Xu and Gordon, 2003). For example, commensal bacteria are required for the complete development of Peyer's patches, the lamina propria, and the intraepithelial spaces, all three of the main immune elements found in the intestine (Duncan and Edberg, 1995; Falk et al., 1998). The intestinal tissue morphology is also altered by commensal colonization such that the villi of the small intestine are longer and the crypts are shorter. In addition, studies in germ-free mice have revealed that the commensal bacteria induce angiogenesis, thereby contributing to the development of the complex vascular beds found just underneath the mucosal surface (Stappenbeck et al., 2002). Furthermore, it has been found that intestinal commensal bacteria contribute to intestinal epithelial cell homeostasis through TLR recognition pathways providing protection from epithelial cell injury (Rakoff-Nahoum et al., 2004). Finally, a state of "controlled" inflammation that normally exists in the intestine has been attributed to both the quality and quantity of intestinal commensal microorganisms (Cebra, 1999; Chadwick and Anderson, 1992).
Full-text · Article · Feb 2013 · Cellular Microbiology