Are Hertwig’s epithelial root sheath cells necessary for periodontal formation by dental follicle cells?

Publication date: October 2018

Source: Archives of Oral Biology, Volume 94

Author(s): Yongwen Guo, Weihua Guo, Jie Chen, Guoqing Chen, Weidong Tian, Ding Bai

Abstract

Objective

The role of Hertwig's epithelial root sheath (HERS) cells in periodontal formation has been controversial. This study aimed to further clarify whether HERS cells participate in formation of the periodontium, and the necessity of HERS cells in differentiation of dental follicle cells (DFCs) for periodontal regeneration.

Design

HERS cells and DFCs were isolated and identified from post-natal 7-day Sprauge-Dawley rats. In vitro, direct co-culture of HERS cells and DFCs as well as the individual culture of HERS and DFCs were performed and followed by alizarin red staining and the quantitative real-time polymerase chain reaction analysis. For in vivo evaluation, the inactivated dentin matrix (iTDM) was fabricated. HERS cells and DFCs were seeded in combination or alone on iTDM and then transplanted into the rat omentum. Scanning electron microscope and further histological analysis were carried out.

Results

In vitro, mineral-like nodules were found in the culture of HERS cells alone or HERS + DFCs either by alizarin red staining or scanning electronic microscope. The mineralization and fiber-forming relevant mRNA expressions, such as bone sialoprotein, osteopontin, collagen I and collagen III in HERS + DFCs were significantly higher than that of the HERS or DFCs alone group. After transplantation in vivo, cementum and periodontal ligament-like tissues were formed in groups of HERS + DFCs and HERS alone, while no evident hard tissues and attached fibers were found in DFCs alone.

Conclusions

Hertwig's epithelial root sheath cells directly participate in the formation of the periodontium, and they are essential for the differentiation of dental follicle cells to form periodontal structures. The combination use of Hertwig's epithelial root sheath cells and dental follicle cells is a promising approach for periodontal regeneration.

https://ift.tt/2IU9Fmg

Inhibitory effects of tea catechin epigallocatechin-3-gallate against biofilms formed from Streptococcus mutans and a probiotic lactobacillus strain

Publication date: October 2018

Source: Archives of Oral Biology, Volume 94

Author(s): Ching-Yi Wu, Tzu-Yi Su, Mao-Yu Wang, Shue-Fen Yang, Kwei Mar, Shan-Ling Hung

Abstract

Objective

Effects of tea catechin epigallocatechin-3-gallate (EGCG) against biofilm formation by Streptococcus mutans and probiotic Lactobacillus casei in Yakult^® (LcY) were examined.

Design

Biofilms were formed by S. mutans alone (Sm) and co-culture of S. mutans and LcY (Sm + LcY) in the absence or presence of EGCG. The biomass of biofilms, which were sonicated or not, was measured by the crystal violet assay. Biofilm morphology was observed by scanning electron microscopy. Bacterial viability and extracellular polysaccharides were determined by SYTO9/propidium iodide and dextran-conjugated fluorescein staining, respectively, and confocal microscopy. Gene expression of glucosyltransferase was determined by quantitative polymerase chain reaction.

Results

While 250 μg/ml EGCG significantly decreased the biomass and acid production of Sm biofilms, 500 μg/ml EGCG was required to inhibit Sm + LcY biofilm formation and acid production. EGCG decreased the amount of live bacteria present in both Sm and Sm + LcY biofilms. The level of dead bacteria in Sm + LcY biofilms was higher than in Sm biofilms when formed in the presence of 250 μg/ml EGCG. EGCG decreased levels of extracellular polysaccharides in Sm and Sm + LcY biofilms. The extent of biofilm removal by sonication was not different between Sm and Sm+LcY biofilms formed in the absence or presence of 62.5 or 125 μg/ml EGCG. The level of Sm gtfB and gtfD expression in Sm + LcY biofilms was higher than those in the Sm biofilms when formed in the presence of EGCG at 250 μg/ml.

Conclusion

The results indicated that LcY might interfere the inhibitory effects of EGCG against biofilm formation by S. mutans.

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Identification of major matrix metalloproteinase-20 proteolytic processing products of murine amelogenin and tyrosine-rich amelogenin peptide using a nuclear magnetic resonance spectroscopy based method

Publication date: September 2018

Source: Archives of Oral Biology, Volume 93

Author(s): Garry W. Buchko, Rajith Jayasinha Arachchige, Jinhui Tao, Barbara J. Tarasevich, Wendy J. Shaw

Abstract

Objective

The aim of this study was to identify major matrix metalloproteinase-20 (MMP20) proteolytic processing products of amelogenin over time and determine if the tyrosine-rich amelogenin peptide (TRAP) was a substrate of MMP20.

Design

Recombinant¹⁵N-labeled murine amelogenin and ¹³C,¹⁵N-labeled TRAP were incubated with MMP20 under conditions where amelogenin self-assembles into nanospheres. Digestion products were fractionated by reverse-phase high-performance liquid chromatography at various time points. Product identification took advantage of the intrinsic disorder property of amelogenin that results in little change to its fingerprint ¹H-¹⁵N heteronuclear single-quantum coherence nuclear magnetic resonance spectrum in 2% acetic acid upon removing parts of the protein, allowing cleavage site identification by observing which amide cross peaks disappear.

Results

The primary product in five out of the six major reverse-phase high-performance liquid chromatography bands generated after a 24 h incubation of murine amelogenin with MMP20 were: S55-L163, P2-L147, P2-E162, P2-A167, and P2-R176. After 72 h these products were replaced with five major reverse-phase high-performance liquid chromatography bands containing: L46-A170, P2-S152, P2-F151, P2-W45, and short N-terminal peptides. TRAP was completely digested by MMP20 into multiple small peptides with the initial primary site of cleavage between S16 and Y17.

Conclusions

Identification of the major MMP20 proteolytic products of amelogenin confirm a dynamic process, with sites towards the C-terminus more rapidly attacked than sites near the N-terminus. This observation is consistent with nanosphere models where the C-terminus is exposed and the N-terminus more protected. One previously reported end-product of the MMP20 proteolytic processing of amelogenin, TRAP, is shown to be an in vitro substrate for MMP20.

https://ift.tt/2KQl88h

Effects of citric acid modified with fluoride, nano-hydroxyapatite and casein on eroded enamel

Publication date: September 2018

Source: Archives of Oral Biology, Volume 93

Author(s): Ayşe Dündar, Abdulkadir Şengün, Canan Başlak, Mahmut Kuş

Abstract

Objectives

The aim of this in vitro study was to evaluate the effects of citric acid containing fluoride, nano-hydroxyapatite, and casein on eroded enamel.

Design

The crowns of 120 extracted bovine incisors were embedded in acrylic resin. An enamel window (2 × 3 mm) was created on the surface. Before in vitro pellicle formation samples were eroded in 1% citric acid (pH = 3.2) for 1 h at 36 °C and were randomly classified to eight groups (n = 15) as follows: Positive control: 1% citric acid, Negative control: Distilled water, F1: 0.047 mmol/L sodium fluoride, F2: 0.071 mmol/L sodium fluoride, NHA1: %0.05 Nano-Hydroxyapatite, NHA2: %0.1 Nano-Hydroxyapatite, C1: %0.02 Casein, C2: %0.2 Casein. Erosion cycling was performed three times daily for 3 days. In each cycle, the samples were immersed in 10 mL of control or modified solutions (10 min) and in 10 mL of artificial saliva (60 min). The surface roughness and enamel loss were analyzed by using profilometer, scanning electron microscopy (SEM) and atomic force microscopy techniques (AFM).

Results

Among the groups, the positive control group was found to be having the highest erosive wear. Erosive wear in the F2, NHA2, C1, and C2 groups was not significantly different from the negative control group (p > 0.05). The C1 and C2 groups showed that erosion terminated and minimal tissue recovery occurred on the enamel surface.

Conclusion

Although all modifications reduced further demineralization, the citric acid modification with casein was found to be having a greater impact on dental erosion than the others.

https://ift.tt/2KL6gLw

Responses to spaceflight of mouse mandibular bone and teeth

Publication date: September 2018

Source: Archives of Oral Biology, Volume 93

Author(s): Didem Dagdeviren, Zana Kalajzic, Douglas J. Adams, Ivo Kalajzic, Alan Lurie, Maija I. Mednieks, Arthur R. Hand

Abstract

Objective

To determine if spaceflight and microgravity affect non-weight bearing bones and development and mineralization of teeth, reasoning that combining an organ and a cellular level approach can lead to greater insights about these effects.

Design

Mandibles and incisors of mice flown on the US STS-135 space shuttle mission and the Russian Bion-M1 satellite were studied using micro-computed tomography and immunohistochemistry. Ground controls were mice housed in standard vivarium cages and flight habitats.

Results

Incisor length was greater in the 13-day STS-135 flight mice than in either control group. Initial incisor mineralization occurred more posteriorly, and incisor, enamel and dentin volumes and enamel and dentin thicknesses were greater in the 30-day Bion-M1 flight and habitat control mice than in vivarium control mice. Mandibular bone volume (BV) was increased in STS-135 flight and habitat groups and decreased in Bion-M1 flight and habitat groups compared to vivarium controls. No significant histological alterations occurred, but changes were seen in the bone and tooth proteins dentin sialoprotein, amelogenin and the type II regulatory subunit of protein kinase A. The percentage of sclerostin positive osteocytes was greatest in flight mice, and greater in STS-135 flight and habitat control mice than in the corresponding Bion-M1 groups. TRAP staining, representing osteoclastic bone remodeling, differed between the two flights and corresponded with changes in BV.

Interpretation of the findings was limited by a small number of flight mice, different sex and ages of the mice in the two missions, and different habitats and diets.

Conclusions

Microgravity has measurable effects on mandibular bone physiology and incisor development and mineralization. The results also showed that the habitat had an effect either in flight or ground control samples, as demonstrated by the changes in BV and apparent slowing of incisor eruption. Therefore, developing appropriate habitats is critical for future spaceflight missions.

https://ift.tt/2IXsAfY

Serotype a of Aggregatibacter actinomycetemcomitans down-regulates the increased serotype b-induced cytokine and chemokine production in dendritic cells

Publication date: September 2018

Source: Archives of Oral Biology, Volume 93

Author(s): Leticia Rojas, Samanta Melgar-Rodríguez, Jaime Díaz-Zúñiga, Carla Alvarez, Gustavo Monasterio, Carolina Rojas, Paola Carvajal, Rolando Vernal

Abstract

Objective

In Aggregatibacter actinomycetemcomitans, different serotypes have been described based on LPS antigenicity. Mixed infection with the different A. actinomycetemcomitans serotypes is frequent in periodontitis patients; accordingly, the role of this bacterial species in the pathogenesis of periodontitis may differ depending whether patients or periodontal lesions harbour one or more of the A. actinomycetemcomitans serotypes. We hypothesized that different combinations of these serotypes could be associated with distinct host responses and hence different inflammatory patterns. This investigation was aimed to assess whether the increased immuno-stimulatory potential attributed to the serotype b of A. actinomycetemcomitans on immune cells is able to be modified during co-infection with other A. actinomycetemcomitans serotypes.

Methods

Dendritic cells (DCs) were obtained from healthy subjects and stimulated with the different A. actinomycetemcomitans serotypes or their purified LPS using the following stimulatory conditions: serotype a, b, or c, and the combinations a+b, a+c, b+c, or a+b+c. The cytokine, CCR, and CCL levels were quantified by qPCR and ELISA.

Results

Higher levels of cytokines, CCRs, and CCLs were induced when DCs were stimulated with the serotype b of A. actinomycetemcomitans compared with the same cells stimulated with the other serotypes. When DCs were co-infected, these levels decreased in comparison with the serotype b-stimulation alone, in particular when the serotype a was present in the mixed infection.

Conclusions

The increased immuno-stimulatory potential attributed to the serotype b was modified when DCs were co-infected with other A. actinomycetemcomitans serotypes, in particular, when the serotype a was present, the DC response diminished.

https://ift.tt/2KMea7b

N-acetyl cysteine inhibits lipopolysaccharide-mediated induction of interleukin-6 synthesis in MC3T3-E1 cells through the NF-kB signaling pathway

Publication date: September 2018

Source: Archives of Oral Biology, Volume 93

Author(s): Ling Guo, Hui Zhang, Wangyang Li, Danting Zhan, Min Wang

Abstract

Background

Interleukin-6 (IL-6) is a potent stimulator of osteoclastic activity. Lipopolysaccharide (LPS) has been shown to regulate the expression of potent inflammatory factors, including TNF-α and IL-6. Currently, effective therapeutic treatments for bacteria-caused bone destruction are limited. N-acetyl cysteine (NAC) is an antioxidant small molecule that possibly modulates osteoblastic differentiation. However, whether NAC can affect the LPS-mediated reduction of IL-6 synthesis in MC3T3-E1 cells is still unknown.

Aims

The aim of this study was to investigate the role of NAC in the LPS -mediated reduction of IL-6 synthesis by MC3T3-E1 cells and to explore the underlying molecular mechanisms. In addition, we aimed to determine the involvement of the NF-kB pathway in any changes in IL-6 expression observed in response to LPS and NAC.

Methods

MC3T3-E1 cells (ATCC, CRL-2593) were cultured in α-minimum essential medium. Cells were stimulated using NAC or LPS at various concentrations. Cell proliferation was observed at multiple time points using a cell counting kit 8 (CCK-8). IL-6 mRNA expression and protein synthesis were determined using quantitative polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay analyses. NF-kB mRNA expression and protein synthesis was determined using qPCR and Western blots analyses.

Results

The results demonstrate that LPS induced IL-6 and NF-kB mRNA expression and protein synthesis in the cultured MC3T3-E1 cells. However, these effects were abolished following pre-treatment with NAC. Pretreatment with NAC (1 mmol/l) or BAY11-7082 (10 μmol/l) both significantly inhibited the NF-kB activity induced by LPS.

Conclusion

NAC inhibits the LPS-mediated induction of IL-6 synthesis in MC3T3-E1 cells through the NF-kB pathway.

https://ift.tt/2IXk14N