[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 healthcare burden worldwide, owing to the formation of robust biofilms against common antifungals. Recent evidence shows that multidrug-tolerant persisters critically account for biofilm recalcitrance, whereas their underlying biological mechanisms are poorly understood. Here, we firstly investigated the phenotypic characteristics of Candida biofilm persisters under consecutive harsh treatments of amphotericin B. The prolonged treatments effectively killed the majority 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 investigating 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, tricarboxylic acid cycle and protein synthesis were markedly down-regulated, indicating that the major metabolic activities are subdued in the persisters. It is noteworthy that certain metabolic pathways could be activated, such as the glyoxylate cycle with significantly increased levels of isocitrate lyase and malate synthase. Moreover, a number of important proteins responsible for Candida growth, virulence and stress response were greatly up-regulated. Interestingly, the persisters were tolerant to oxidative stress, despite of highly induced intracellular superoxide. The current findings suggest that delicate metabolic control and coordinated stress response may play a crucial role in mediating the survival and antifungal tolerance of Candida biofilm persisters.
[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.
Journal of endodontics 02/2015; DOI:10.1016/j.joen.2014.12.017 · 3.38 Impact Factor
[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.
Tissue Engineering Part A 11/2014; DOI:10.1089/ten.TEA.2014.0058 · 4.64 Impact Factor
[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.
Tissue Engineering Part A 09/2014; DOI:10.1089/ten.TEA.2014.0154 · 4.64 Impact Factor
[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.
Journal of Dental Research 09/2014; DOI:10.1177/0022034514550040 · 4.14 Impact Factor
[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.
PLoS ONE 08/2014; 9(8):e103234. DOI:10.1371/journal.pone.0103234 · 3.23 Impact Factor
[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.
[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: Background
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.
This study systematically investigated the effects of P. gingivalis LPS1435/1449 and LPS1690 on the expression of TLR2 and TLR4 signal transduction and the activation of pro-inflammatory cytokines IL-6 and IL-8 in human gingival fibroblasts (HGFs). We found that LPS1435/1449 and LPS1690 differentially modulated TLR2 and TLR4 expression. NF-κB pathway was significantly activated by LPS1690 but not by LPS1435/1449. In addition, LPS1690 induced significant expression of NF-κB and p38 MPAK pathways-related genes, such as NFKBIA, NFKB1, IKBKB, MAP2K4 and MAPK8. Notably, the pro-inflammatory genes including GM-CSF, CXCL10, G-CSF, IL-6, IL-8 and CCL2 were significantly upregulated by LPS1690 while down-regulated by LPS1435/1449. Blocking assays confirmed that TLR4-mediated NF-κB signaling was vital in LPS1690-induced expression of IL-6 and IL-8 in HGFs.
The present study suggests that the tetra- and penta-acylated lipid A structures of P. gingivalis LPS differentially activate TLR4-mediated NF-κB signaling pathway, and significantly modulate the expression of IL-6 and IL-8 in HGFs. The ability to alter the lipid A structure of LPS could be one of the strategies carried-out by P. gingivalis to evade innate host defense in gingival tissues, thereby contributing to periodontal pathogenesis.
PLoS ONE 03/2013; 8(3):e58496. DOI:10.1371/journal.pone.0058496 · 3.23 Impact Factor
[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).
[Show abstract][Hide abstract] ABSTRACT: Periodontal (gum) disease is one of the main global oral health burdens and severe periodontal disease (periodontitis) is a leading cause of tooth loss in adults globally. It also increases the risk of cardiovascular disease and diabetes mellitus. Porphyromonas gingivalis lipopolysaccharide (LPS) is a key virulent attribute that significantly contributes to periodontal pathogenesis. Baicalin is a flavonoid from Scutellaria radix, an herb commonly used in traditional Chinese medicine for treating inflammatory diseases. The present study examined the modulatory effect of baicalin on P. gingivalis LPS-induced expression of IL-6 and IL-8 in human oral keratinocytes (HOKs). Cells were pre-treated with baicalin (0-80 µM) for 24 h, and subsequently treated with P. gingivalis LPS at 10 µg/ml with or without baicalin for 3 h. IL-6 and IL-8 transcripts and proteins were detected by real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The expression of nuclear factor-κB (NF-κB), p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins was analyzed by western blot. A panel of genes related to toll-like receptor (TLR) signaling was examined by PCR array. We found that baicalin significantly downregulated P. gingivalis LPS-stimulated expression of IL-6 and IL-8, and inhibited P. gingivalis LPS-activated NF-κB, p38 MAPK and JNK. Furthermore, baicalin markedly downregulated P. gingivalis LPS-induced expression of genes associated with TLR signaling. In conclusion, the present study shows that baicalin may significantly downregulate P. gingivalis LPS-upregulated expression of IL-6 and IL-8 in HOKs via negative regulation of TLR signaling.
PLoS ONE 12/2012; 7(12):e51008. DOI:10.1371/journal.pone.0051008 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: LPS-binding protein (LBP) functions as a crucial molecule in innate immune responses to bacterial challenge. Our study has shown the expression of LBP in human gingiva and its significant association with periodontal health and disease. Porphyromonas gingivalis is a key pathogen of periodontal disease. P. gingivalis LPS as a main virulence factor is strongly involved in periodontal pathogenesis and it displays a significant lipid A structural heterogeneity. Currently, it remains unknown whether, and to what extent, the lipid A structural heterogeneity of P. gingivalis LPS affects LBP expression. The present study investigated the expression profile of LBP in human oral keratinocytes (HOKs) stimulated by two isoforms of P. gingivalis LPS [tetra- (LPS(1435/1449)) and penta-acylated (LPS(1690))] and Escherichia coli LPS, and the involvement of TLRs in LBP expression. The results showed that the expression of LBP mRNA and peptide was significantly up-regulated by P. gingivalis LPS(1690) and E. coli LPS, while P. gingivalis LPS(1435/1449) did not affect LBP expression. Blocking assay and siRNA gene silencing revealed that P. gingivalis LPS(1690)-induced LBP expression was through both TLR2 and TLR4. This in vitro study demonstrates that P. gingivalis LPS with a lipid A structural heterogeneity differentially modulates LBP expression in HOKs.
[Show abstract][Hide abstract] ABSTRACT: Abstract – Background: Hank’s balanced salt solution (HBSS) and milk have gained wide acceptance as storage media for avulsed tooth. However, the effect of the media and storage time on the periodontal ligament (PDL) cells involvement in the development of root resorption is still unclear. The purpose of this study was to evaluate whether precultured PDL cells in HBSS, milk, or modified Eagle’s medium alpha (α-MEM) would affect osteoclastogenesis.
Materials and methods: PDL cells were precultured in HBSS, milk, or α-MEM for 1 h or 6 h before being co-cultured with RAW 264.7 cells for an additional 3 days for mRNA analysis and 11 days for osteoclastogenesis assay.
Results: Cyclooxygenase-2 (COX-2) mRNA was detected immediately in PDL cells precultured in the three storage media. The expression was up-regulated markedly in all co-cultures when compared with RAW cells alone. As a result of the co-culture, interleukin-1β (IL-1β) expression was detectable in both PDL and RAW cells. TRAP+ multinucleated, osteoclast-like cells developed in all co-cultures; the number of TRAP+ cells was highest (P < 0.05) in the co-cultures that PDL cells precultured in milk for 6 h. The mRNA level of receptor activator of nuclear factor-kappa B ligand (RANKL) was not detected in PDL cells. Osteoprotegerin (OPG) mRNA expression reduced with increased preculture time, but the difference was not significant (P > 0.05).
Conclusions: PDL cells kept in the three storage media led to TRAP+ multinucleated, osteoclast-like cells formation via RANKL-independent signaling. The ability to induce osteoclastogenesis may be considered as one of the factors to evaluate the ability of storage medium to maintain PDL viability after tooth avulsion.
[Show abstract][Hide abstract] ABSTRACT: Dental pulp stem cells (DPSCs) have received much attention as a promising population of stem cells in regenerative endodontics. Securing a good blood supply during regeneration is a challenging task because of the constricted apical canal opening, which allows only a limited blood supply. The aim of this study was to investigate any potential synergistic effects of dental pulp stem cells and endothelial cells (ECs) on osteo-/odontogenic and angiogenic differentiation in vitro.
Different ratios of DPSCs and ECs were cultured in direct contact using optimized medium for coculture. The 70% confluent cocultures were incubated in the osteo-/odontogenic differentiation medium for up to 3 weeks. Alkaline phosphatase (ALP) activity, the expression levels of ALP, bone sialoprotein (BSP), dentin sialophosphoprotein (DSPP) genes, and alizarin red staining for mineralization at different time points were analyzed. The tubular network formation on Matrigel and the gene expression levels of CD117, VEGF, CD34, and Flk-1 were used as assays to analyze angiogenesis.
The quantification of ALP in DPSC:EC cocultures revealed a greater ALP activity compared with DPSC-alone cultures. At all the time points, 1:1 cultures showed a significantly greater ALP activity than that of DPSC-alone cultures. Alizarin red staining and quantification revealed a much greater amount of calcification in the 1:1 and 1:5 cocultures compared with other cultures (P < .01). The expression levels of ALP, BSP, and DSPP genes further confirmed the greater osteo-/odontogenic differentiation in cocultures compared with those of DPSC-alone cultures. Matrigel assay showed that the addition of DPSCs stabilized preexisting vessel-like structures formed by ECs and increased the longevity of them.
Direct coculture of DPSCs and ECs enhances the in vitro differentiation toward osteo-/odontogenic and angiogenic phenotypes.
Journal of endodontics 04/2012; 38(4):454-63. DOI:10.1016/j.joen.2011.12.024 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Biofilms are surface-attached, matrix-encased, structured microbial communities which display phenotypic features that are dramatically different from those of their free-floating, or planktonic, counterparts. Biofilms seem to be the preferred mode of growth of microorganisms in nature, and at least 65% of all human infections are associated with biofilms. The most notable and clinically relevant property of biofilms is their greater resistance to antimicrobials compared with their planktonic counterparts. Although both bacterial and fungal biofilms display this phenotypic feature, the exact mechanisms underlying their increased drug resistance are yet to be determined. Advances in proteomics techniques during the past decade have facilitated in-depth analysis of the possible mechanisms underpinning increased drug resistance in biofilms. These studies have demonstrated the ability of proteomics techniques to unravel new targets for combating microbial biofilms. In this review, we discuss the putative drug resistance mechanisms of microbial biofilms that have been uncovered by proteomics and critically evaluate the possible contribution of the new knowledge to future development in the field. We also summarize strategic uses of novel proteomics technologies in studies related to drug resistance mechanisms of microbial biofilms.
[Show abstract][Hide abstract] ABSTRACT: Dogs are commonly used animal models for regenerative endodontics research. Although several studies have used stem cells isolated from dog teeth to investigate the dentin/pulp regeneration in vivo, less attention has been paid for the characterization of these cells. Therefore, this study aimed to characterize the dental pulp stem cells isolated from dog teeth (cDPSCs) in order to further define the dog as an animal model for regenerative endodontics.
Stem cells were isolated from freshly extracted premolars of 10-month-old Beagles. The isolated cells were investigated for their stem cell properties by analysis of their clonogenic and growth characteristics; expression of mesenchymal stem cell markers; and evaluation of their osteo/odontogenic, adipogenic, and neurogenic potential.
A colony formation assay showed the existence of a clonogenic cell population in cDPSCs isolated. The growth curves revealed a higher proliferation rate of cDPSCs compared with hBMMSCs. cDPSCs expressed mesenchymal stem cell markers STRO-1, CD146, and Nanog. However, they were negative for CD73, CD105, and CD45. cDPSCs were able to differentiate into odontoblast-like cells as shown by increased alkaline phosphatase activity, dentin sialoprotein expression, and formation of mineralized nodules. cDPSCs also showed the neurogenic and adipogenic differentiation potential at a lower level compared with those of hDPSCs and hBMMSCs.
The results of this study confirmed the stem cell properties of cDPSCs at a comparable level to those of hDPSCs and hBMMSCs. Overall, the data presented in this study provide evidence in supportive of using cDPSCs and dogs as an animal model in dental tissue engineering via stem cell-based approaches.
Journal of endodontics 08/2011; 37(8):1074-80. DOI:10.1016/j.joen.2011.04.004 · 3.38 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Porphyromonas gingivalis lipopolysaccharide (LPS) displays a significant amount of structural heterogeneity, containing both tetra- (LPS(1435/1449) ) and penta-acylated (LPS(1690) ) lipid A structures. This study investigated the effects of the two isoforms of P. gingivalis LPS on the expression of IL-6, IL-8 and TNF-α in human gingival fibroblasts (HGFs).
HGFs were stimulated with P. gingivalis LPS(1435/1449) and LPS(1690) in both dose- (1 ng-10 μg/ml) and time-dependent (2-48 h) experiments. Total RNA and protein were extracted and used for analysis of the IL-6, IL-8 and TNF-α transcripts as well as IL-6 and IL-8 proteins, by quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.
P. gingivalis LPS(1690) significantly up-regulated the mRNA and protein expression of IL-6 and IL-8, whereas P. gingivalis LPS(1435/1449) did not induce significant host response. The expression levels of IL-6 and IL-8 up-regulated by P. gingivalis LPS(1690) continuously increased with time course. In contrast, TNF-α transcript expression was up-regulated promptly by P. gingivalis LPS(1690) after 2 h of stimulation and gradually declined afterwards.
This study suggests that P. gingivalis LPS heterogeneity may differentially modulate the pro-inflammatory cytokine expression in HGFs, which may contribute to periodontal pathogenesis.
[Show abstract][Hide abstract] ABSTRACT: Interleukin-6 (IL-6) is a potent stimulator of osteoclastic bone resorption. Osteoblast secretion of IL-6 plays an important role in the regulation of bone metabolism. Angiotensin II (Ang II) has been shown to regulate the expression of potent inflammatory factors, including MCP-1 and IL-6, by stimulating endothelial cells, vascular smooth muscle cells (VSMC) and monocytes. However, of the mechanism by which Ang II regulates IL-6 expression in osteoblasts is unknown.
The present study was designed to investigate the effect of Ang II on IL-6 expression in osteoblasts isolated from mice. The receptor(s) required and the potential role of extracellular signal-regulated kinase 1/2 (ERK1/2) activation in Ang II-induced IL-6 synthesis was also examined in these cells.
The osteoblasts were isolated from the calvaria of mice and cultured in α-MEM medium. IL-6 mRNA expression and protein synthesis was determined by qPCR and ELISA analyses. ERK1/2 kinase activation was determined by western blot.
The results indicate that Ang II induced IL-6 mRNA expression and protein synthesis in cultured osteoblasts. However, these effects were abolished by pre-treatment with Ang II type 1 (AT1) receptor antagonist, losartan, and the ERK1/2 inhibitor, U0126, inhibited Ang II-mediated IL-6 expression and the phosphorylation of ERK1/2.
Ang II induces the synthesis of IL-6 in osteoblasts through activation of the ERK1/2 pathway via the AT1 receptor.