The Proteomes of Oral Cells Change during Co-Cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens.

BACKGROUND: Changes in the proteome of oral cells during periodontitis have rarely been investigated. This lack of information is partially attributed to the lack of human cell lines derived from the oral cavity for in vitro research. The objective of the present study was to create cell lines from relevant oral tissues and compare protein expression in cells cultured alone and in cells co-cultivated with periodontitis-associated bacterial strains. METHODS: We established human cell lines of gingival keratinocytes, osteoblastic lineage cells from the alveolar bone, periodontal ligament fibroblasts, and cementum cells. Using state-of-the-art label-free mass spectrometry, we investigated changes in the proteomes of these cells after co-cultivation with Aggregatibacter actinomycetemcomitans and Eikenella corrodens for 48 h. RESULTS: Gingival keratinocytes, representing ectodermal cells, exhibited decreased expression of specific keratins, basement membrane components, and cell-cell contact proteins after cultivation with the bacterial strains. Mesodermal lineage cells generally exhibited similar proteomes after co-cultivation with bacteria; in particular, collagens and integrins were expressed at higher levels. CONCLUSIONS: The results of the present study will help us elucidate the cellular mechanisms of periodontitis. Although co-cultivation with two periodontitis-associated bacterial strains significantly altered the proteomes of oral cells, future research is needed to examine the effects of complex biofilms mimicking in vivo conditions.

Proliferation and Morphological Assessment of Human Periodontal Ligament Fibroblast towards Bovine Pericardium Membranes: An In Vitro Study.

Over the past decade regenerative branches of dentistry have taken on more and more importance, resulting in the development of performing scaffold materials. These should induce cell adhesion, support, and guide the tissues' growth. Among the developed materials, we can include resorbable or non-membranes. The purpose of this study was to investigate the proliferation abilities and the attachment of human periodontal ligament fibroblasts (HPLIFs) over two bovine pericardium membranes with different thicknesses, 0.2 mm and 0.4 mm, respectively. These membranes have been decellularized by the manufacturer, preserving the three-dimensional collagen's structure. The HPLFs were cultured in standard conditions and exposed to the tested materials. XTT was performed to assess cell proliferation, while light microscopy (LM) and scanning electron microscopy (SEM) observations assessed fibroblast morphology at different times (T1, T2, and T3). Proliferation assays have shown a statistically significant difference in growth at T1 (p < 0.05) in the cells cultured with a thicker membrane compared to the thinner one. LM analysis showed healthy fibroblasts in contact with the membranes, appearing larger and with a polygonal shape. SEM observation demonstrated thickening of the fibroblasts which continued to adhere to the membrane's surface, with enlarged polygonal shape and developed filipodia and lamellipodia. These results showed a similar cell behavior over the two bovine pericardium membranes, demonstrating a cellular migration along and within the layers of the membrane, binding with membrane fibers by means of filopodial extensions. Knowledge of the effects of the collagen membranes derived from bovine pericardium on cellular behavior will help clinicians choose the type of scaffolds according to the required clinical situation.

MMP and TIMP production in periodontal ligament fibroblasts stimulated by Prevotella nigrescens lipopolysaccharide / 대한치과보존학회지

The purpose of this study was to monitor the secretion of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) by human periodontal ligament (PDL) fibroblasts stimulated with Prevotella nigrescens lipopolysaccharide (LPS), and to examine the effect of calcium hydroxide treatment on P. nigrescens LPS. LPS was extracted and purified from anaerobically cultured P. nigrescens. PDL fibroblasts were stimulated by the LPS (0, 0.1, 1, 10 ug/ml) or LPS (10 ug/ml) pretreated with 12.5 mg/ml of Ca(OH)2 for 3 days, for various periods of time (12, 24, 48 h). Immunoprecipitation were performed for protein level analysis of MMP-1, MMP-2 and TIMP-1. Total RNA was isolated and real-time quantitative polymerase chain reaction (PCR) was performed for quantification of MMP-1 mRNA. According to this study, the results were as follows: 1. The production of MMP-1 by stimulation with P. nigrescens LPS increased in time-dependent manner, and showed maximum value at 48 h in both protein and mRNA level. But there was no dose-dependent increase. 2. MMP-2 production time-dependently increased when stimulated with 1 and 10 ug/ml LPS, but there was no dose-dependent increase. 3. TIMP-1 production increased to 24 h, but decreased at 48 h. It increased when stimulated with 0.1 and 1 ug/ml LPS, but suppressed at 10 ug/ml. 4. P. nigrescens LPS pretreated with Ca(OH)2 markedly downregulated MMP-1 gene expression.

Analysis of PDL Fibroblast Change During Mechanical Stimuli in the Rats / 대한치주과학회지

Periodontal disease is characterized by inflammation and subsequent loss and/or damage to tooth-supporting tissues such as bone, cementum, and periodontal ligament.Periodontal ligament and cementum are the key tissues in the initial process of regeneration following periodontal disease.Therefore, studies on cementoblasts, which form cementum are emphasized. It is still unclear which cells cementoblast differentiate from.This study was conducted under the hypothesis that PDL fibroblast can differentiate into either cementoblast or osteoblast depending on the conditions of surrounding tissue.Clinically, with excessive traction force of orthodontic appliances or excessive occlusion hypercementosis is observed, and this has been confirmed histologically.Consequently, activation of cementoblast can be expected in rats when mechanical stimuli are given to PDL fibroblast.Therefore, the purpose of this article is to prove that PDL fibroblast differentiates into cementoblast in rats under mechanical stimuli using histologic and molecular methods. In this study, twenty rats were given hard diet.Ten of them were sacrificed after 1 week, and the others were sacrificed after two weeks.Slides were made from tooth specimen, and they were studied under the microscope. In addition, PDL fibroblast and cementum from the extracted teeth were analyzed with Northern blotting. In histologic examination, as time passed, PDL fibroblast migrated to the dentin side, differentiated into cementoblast, and formed new cementum.In Northern blotting, it was found that mRNA expression of cementoblast-specific proteins such as BSP, OC, OPN, and type I collagen were more prominent in rats sacrificed after 2 weeks of hard-diet than rats sacrificed after 1 week. From these findings we can conclude that PDL fibroblast can differentiate into cementoblast under mechanical stimuli.We think that 'Rat Models' used in this study will be beneficial to future studies regarding cementoblast.