Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament

Shimonishi M, Takahashi I, Terao F, Komatsu M, Kikuchi M. Induction of MMP‐2 at the interface between epithelial cells and fibroblasts from human periodontal ligament. J Periodont Res 2010; 45: 309–316. © 2009 John Wiley & Sons A/S Background and Objective: MMP‐2 can degrade type IV collagen and MMP‐14 can activate pro MMP‐2. The present study was undertaken to examine the expression of MMP‐2 and MMP‐14 with respect to interaction between the cells of the epithelial rests of Malassez and fibroblasts from human periodontal ligament. Material and Methods: Explants of human periodontal ligament tissues produced outgrowths containing both putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts after incubation in a modified serum‐free medium. The distribution and expression of MMP‐2 and MMP‐14 were analysed using immunohistochemistry, in situ hybridization and RT‐PCR analysis. The conditioned media and cell extracts were collected for western blot analysis for MMP‐2. Results: Putative epithelial rests of Malassez cells at the interface between the cells of the epithelial rests of Malassez and fibroblasts expressed MMP‐2 and MMP‐14 strongly. However, in situ hybridization analysis revealed that human periodontal ligament fibroblasts expressed MMP‐2 mRNA while putative epithelial rests of Malassez cells expressed MMP‐14 mRNA at the interface. The RT‐PCR analysis showed that the expression of MMP‐2 mRNA was significantly higher when putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts were cultured together than when cultured alone. Western blot analysis showed that the active form of MMP‐2 was detected at higher levels in the conditioned medium of the co‐cultured cells. Conclusion: These findings indicate that putative epithelial rests of Malassez cells stimulate the production of MMP‐2 in human periodontal ligament fibroblasts. Up‐regulated proMMP‐2 bound by MMP‐14 expressed in epithelial rests of Malassez cells can degrade matrix molecules, such as type IV collagen, in the basal membrane between putative epithelial rests of Malassez cells and human periodontal ligament fibroblasts.     

Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction with Porphyromonas gingivalis

Scheres N, Laine ML, Sipos PM, Bosch‐Tijhof CJ, Crielaard W, de Vries TJ, Everts V. Periodontal ligament and gingival fibroblasts from periodontitis patients are more active in interaction withPorphyromonas gingivalis. J Periodont Res 2011; 46: 407–416. © 2011 John Wiley & Sons A/S Background and Objective: Inflammatory responses of host cells to oral pathogenic bacteria, such as Porphyromonas gingivalis, are crucial in the development of periodontitis. Host cells, such as periodontal ligament and gingival fibroblasts, from periodontitis patients may respond to P. gingivalis in a different manner compared with cells from healthy persons. The aim of this study was to investigate inflammatory responses to viable P. gingivalis by periodontal ligament and gingival fibroblasts from periodontitis patients and healthy control subjects. Material and Methods: Primary periodontal ligament and gingival fibroblasts from periodontitis patients (n = 14) and healthy control subjects (n = 8) were challenged in vitro with viable P. gingivalis. Gene expression of Toll‐like receptors (TLRs) 1, 2, 4, 6, 7 and 9, CD14, nuclear factor‐κB1 and its putative inhibitor NF‐κB inhibitor‐like protein 1, and of interleukin‐1β, interleukin‐6, interleukin‐8, tumour necrosis factor‐α, monocyte chemotactic protein‐1 and regulated upon activation, normal T‐cel expressed, and secreted, were assessed by real‐time PCR. Results: Periodontal ligament fibroblasts from periodontitis patients had a higher mRNA expression of TLR1, TLR4, TLR7 and CD14, and a lower expression of NFKBIL1, both before and after P. gingivalis challenge. In contrast, gingival fibroblasts from periodontitis patients had stronger induction of TLR1, TLR2 and TLR7 by P. gingivalis. Cytokine responses were not different between patients and control subjects. Interestingly, periodontal ligament, but not gingival, fibroblasts from P. gingivalis culture‐positive persons responded more strongly to P. gingivalis than periodontal ligament fibroblasts from P. gingivalis‐negative persons. Conclusion: Periodontal ligament and gingival fibroblasts respond to P. gingivalis in a different manner and may play different roles in periodontitis. Both subsets of fibroblasts from patients appear more active in interaction with P. gingivalis. Moreover, periodontal ligament fibroblasts from P. gingivalis‐positive donors are more responsive to an in vitro P. gingivalis challenge.     

Interleukin-1 regulation of procollagenase mRNA and protein in periodontal fibroblasts in vitro

An in vitro model is used to investigate the hypothesis that activated fibroblasts produce collagenolytic activity in inflammatory sites. Interleukin-1, a cytokine present in the gingiva and crevicular fluid of periodontitis patients, has multiple biologic activities including the ability to stimulate collagenase in dermal and synovial fibroblasts. In this report IL-1 is tested on gingival (GF) and periodontal ligament fibroblasts (PLF) for its ability to increase collagenolytic activity and procollagenase mRNA and protein. GF produce a 3- to 7-fold increase in collagenase activity, while PLF collagenase activity is rarely increased above control amounts by IL-1 treatment. In contrast, both cell types demonstrate an increase in procollagenase protein production with IL-1 treatment. RNA from both GF and PLF contain procollagenase mRNA as demonstrated when northern blots of fibroblast total RNA are hybridized with the cDNA for human procollagenase. Treatment with IL-1 increases the steady-state levels of this message in GF by up to 10-fold in 48 hours when measured with dot blot analysis standardized for poly-A RNA. PLF also produce up to 7 times more message at the same dose and time. Since fibroblasts present in the lesion are exposed to inflammatory cell products it is possible that the production of collagenase by these cells could result in the destruction of the periodontal fibrous attachment.     

Biology and pathogenesis of cytomegalovirus in periodontal disease

Human periodontitis is associated with a wide range of bacteria and viruses and with complex innate and adaptive immune responses. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans, Treponema denticola, cytomegalovirus and other herpesviruses are major suspected pathogens of periodontitis, and a combined herpesvirus–bacterial periodontal infection can potentially explain major clinical features of the disease. Cytomegalovirus infects periodontal macrophages and T‐cells and elicits a release of interleukin‐1β and tumor necrosis factor‐α. These proinflammatory cytokines play an important role in the host defense against the virus, but they also have the potential to induce alveolar bone resorption and loss of periodontal ligament. Gingival fibroblasts infected with cytomegalovirus also exhibit diminished collagen production and release of an increased level of matrix metalloproteinases. This article reviews innate and adaptive immunity to cytomegalovirus and suggests that immune responses towards cytomegalovirus can play roles in controlling, as well as in exacerbating, destructive periodontal disease.     

Transcellular invasive mechanisms of Porphyromonas gingivalis in host–parasite interactions

Porphyromonas gingivalis, a well-known periodontal pathogen, expresses a number of virulence factors, including fimbriae and gingipains. In addition, the pathogen utilizes two unique transcellular invasive mechanisms that cause cellular impairment in periodontal tissues; these mechanisms include bacterial entry into periodontal cells and bacterial shooting of outer membrane vesicles (OMVs) into those cells.Gingival epithelial cells function as innate host defense barriers to prevent intrusion by periodontal bacteria. Nevertheless, P. gingivalis can enter these cells and pass through the epithelial barrier into deeper tissues. Fimbriae of P. gingivalis specifically interact with α5β1-integrin of epithelial cells, which induces cellular invagination to internalize the pathogen. Following their entry, intracellular P. gingivalis impairs fundamental cellular functions, while some intracellular bacteria are finally sorted to lytic compartments, including autolysosomes and late endosomes/lysosomes. Furthermore, a considerable number of organisms are sorted into recycling endosomes, leading to bacterial exit from infected cells to neighboring cells, a mechanism of cell-to-cell spread within periodontal tissues.Most gram-negative bacteria, including P. gingivalis, produce OMVs, which serve as bacterial “bullets” for directed intercellular transport of bacterial virulence factors into host cells and tissues. Following entry into the cells, OMVs can use bacterial gingipains to degrade cellular functional components. Although OMVs do not escape from lytic compartments, they survive within lysosomes for more than 24 h, resulting in significant formation of acidified compartments. This review addresses the remarkable transcellular strategies that are used by P. gingivalis to destroy periodontal tissues.     

Th2 cytokines efficiently stimulate periostin production in gingival fibroblasts but periostin does not induce an inflammatory response in gingival epithelial cells

Objectives

This study aims to clarify whether gingival fibroblasts produce periostin in response to Th2 cytokines which are elevated in periodontitis lesion and, if so, whether periostin affects the inflammatory response and matrix-protein metabolism.

Design

Human gingival fibroblasts, periodontal ligament cells and the gingival epithelial cell line epi4 were stimulated with interleukin-4 (IL-4), IL-13, tumour necrosis factor-α (TNF-α) and Porphyromonas gingivalis lipopolysaccharide (LPS). Periostin expression was analysed by real-time polymerase chain-reaction (PCR) and Western blotting. The expression of the IL-4 receptor α-chain was evaluated by immunocytochemistry. The effect of periostin on the production of inflammatory cytokines and the expression of matrix protein-related genes was analysed by real-time PCR and enzyme-linked immunosorbent assay (ELISA).

Results

While IL-4 and IL-13 significantly induced periostin production in gingival fibroblasts and periodontal ligament cells, no effect was observed in epi4 cells. No stimulatory effect of TNF-α or P. gingivalis LPS on the production of periostin was observed. The effect of periostin on the production of inflammatory cytokines was weak in gingival fibroblasts; however, little or no effect was observed on periodontal ligament cells or epi4 cells. No significant effect of periostin on the expression of matrix protein-related genes was found.

Conclusion

The results suggest that gingival fibroblasts may be a source of periostin in periodontitis lesions but periostin has only a limited role either in the inflammatory response or in matrix-protein metabolism. Thus, the role of periostin in the cellular interaction between epithelial and mesenchymal cells in gingiva may be distinct from that of skin.     

Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium

Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues. Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez. No matrilysin expression was seen in the periodontal pocket tissue. In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells. Of 13 anaerobic oral bacterial species tested, F. nucleatum, F. necrophorum, P. endodontalis, and P. denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells. The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1. The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.     

Modulation of host matrix metalloproteinases by bacterial virulence factors relevant in human periodontal diseases

OBJECTIVE: Bacterial pathogens involved in periodontal diseases exert a part of their destructive effect by triggering and inducing host cells to elevate their secretion of matrix metalloproteinases (MMPs). Pathogen-secreted phospholipase (PLC) is one bacterial product that may trigger this host response. The roles of exogenous PLC leading to the release, secretion and expression of MMPs by peripheral blood neutrophils (PMNs), cultured epithelial cells of human gingiva and porcine periodontal ligament were investigated. Also the activities of PLC in the diseased and healthy gingival sulcular fluid (crevicular fluid, GCF) and molecular forms of gelatin-ases present in dental plaque were investigated. MATERIALS AND METHODS GCF, salivary and dental plaque samples were analyzed for PLC and proteinase activities. The abilities of PLC to induce PMNs and oral epithelial cells to release and express their MMPs were examined by specific functional, immunological and molecular biology means. RESULTS: PMN-derived MMPs were found to predominate in periodontitis GCF and plaque, and PLC activities were higher in GCF of adult periodontitis patients than in healthy controls. Purified bacterial PLC (1 mU ml^-I) efficiently induced PMN degranulation. PLC also induced MMP expression in the cultured epithelial cells. The strongest response was seen in MMP-9 and less in MMP-2. The induction was dose-dependent in the range of 0.I-1.0 U ml^-1 PI-PLC, and quiescent cultures were more responsive than proliferating ones. PLC induction of MMPs was polar, with increased levels of MMP-9 in the apical region and increased MMP-2 levels secreted in the basal direction. Northern analysis showed a strong increase in mRNA levels of MMP-9 and a smaller increase for MMP-2 and MMP-I. In the second part of the study we investigated the molecular forms of the released MMPs during periodontitis. In bacterial plaque of periodontitis patients the MMP-9 were found to be converted into lower molecular weight forms. Isolated proteinase from Porphyromonas gingivalis (ATCC 33277) was able to convert human proMMPs to their active forms. CONCLUSION: Bacterial PLC may induce degranulation of PMN MMPs and increase MMP expression in oral epithelial cells. The released proteases can be converted into active form by the proteases of plaque bacteria. Thereby, the pathogenic oral bacteria may indirectly participate in the destruction of periodontal tissues.     

Degradation of collagen-guided tissue regeneration membranes by proteolytic enzymes of Porphyromonas gingivalis and its inhibition by antibacterial agents

Previous studies have shown that whole cells of several periodontal pathogenic bacteria including Porphyromonas gingivalis may degrade the clinically used regeneration membranes Biomend Extend^? and Bio‐Gide^®. Fractionation of P. gingivalis cells revealed that cell membrane‐associated proteases are responsible for the in vitro degradation of the collagen membranes. In the present study, the specific role of extracellular vesicles and the purified Arg‐gingipain enzyme of P. gingivalis in the degradation of three differently cross‐linked collagen membranes (Ossix^?; Bio‐Gide^® and Biomend Extend^?) was examined. In addition, the inhibitory effect of antibacterial agents and antibiotics used in local periodontal therapy on the enzymatic degradation was evaluated. The data presented show that while all tested collagen membranes, are prone to lysis by oral bacterial proteases, cross‐linked membranes are more resistant to proteolysis. Furthermore, therapeutical concentrations of the antibacterial and antibiotic agents chlorhexidine, cetylpyridiniumchloride, minocycline and doxycycline were found to partially inhibit the enzymatic breakdown of the membranes, while metronidazole had no such effect. These results suggest that the presence of P. gingivalis cells, extracellular vesicles and enzymes in the vicinity of regeneration membranes in the periodontium, may change their physical structure and therefore alter their biological properties. Furthermore, the use of cross‐linked collagen membranes and antibacterial agents may significantly inhibit this proteolytic process.     

Secreted adenosine triphosphate from Aggregatibacter actinomycetemcomitans triggers chemokine response

Extracellular ATP (eATP) is an important intercellular signaling molecule secreted by activated immune cells or released by damaged cells. In mammalian cells, a rapid increase of ATP concentration in the extracellular space sends a danger signal, which alerts the immune system of an impending danger, resulting in recruitment and priming of phagocytes. Recent studies show that bacteria also release ATP into the extracellular milieu, suggesting a potential role for eATP in host–microbe interactions. It is currently unknown if any oral bacteria release eATP. As eATP triggers and amplifies innate immunity and inflammation, we hypothesized that eATP secreted from periodontal bacteria may contribute to inflammation in periodontitis. The aims of this study were to determine if periodontal bacteria secrete ATP, and to determine the function of bacterially derived eATP as an inducer of inflammation. Our results showed that Aggregatibacter actinomycetemcomitans, but not Porphyromonas gingivalis, Prevotella intermedia, or Fusobacterium nucleatum, secreted ATP into the culture supernatant. Exposure of periodontal fibroblasts to filter sterilized culture supernatant of A. actinomycetemcomitans induced chemokine expression in an eATP‐dependent manner. This occurred independently of cyclic adenosine monophosphate and phospholipase C, suggesting that ionotrophic P2X receptor is involved in sensing of bacterial eATP. Silencing of P2X7 receptor in periodontal fibroblasts led to a significant reduction in bacterial eATP‐induced chemokine response. Furthermore, bacterial eATP served as a potent chemoattractant for neutrophils and monocytes. Collectively, our findings provide evidence for secreted ATP of A. actinomycetemcomitans as a novel virulence factor contributing to inflammation during periodontal disease.     

Initial characterization of a neutral metalloproteinase, active on native 3/4-collagen fragments, synthesized by ROS 17/2.8 osteoblastic cells, periodontal fibroblasts, and identified in gingival crevicular fluid

Analysis of collagenolytic activity in gingival crevicular fluid (GCF) has revealed the presence of an enzyme capable of fragmenting native 3/4- and 1/4-collagen cleavage products generated by collagenase. An enzyme with similar activity was also identified in media conditioned by fibroblasts from rat periodontal ligament and gingiva, and by rat osteoblastic cells (ROS 17/2.8, 17/2A, 17/2B). In culture, the enzyme was secreted in a latent form that could be activated by organomercurials. For further characterization of this novel enzyme (MMP-V), the osteoblast proteinase was partially purified. ROS 17/2.8 conditioned medium was harvested daily and the 25%-60% sat. ammonium sulfate fraction chromatographed on an AcA 54 gel filtration column. Latent forms of MMP-V (apparent Mr approximately 54 k) and collagenase (Mr approximately 54 k) were resolved from gelatinase (Mr approximately 76 k) and two collagenase inhibitors (Mr approximately 62 k, approximately 36 k). Activated MMP-V degraded native 3/4-collagen fragments from collagen types I and II in a step-wise manner and was active on denatured collagen. MMP-V showed a divalent cation requirement, was active at neutral pH, and was inhibited by collagenase inhibitor and fetal bovine serum, but not by serine, thiol, or carboxyl proteinase inhibitors. These properties indicate that MMP-V is a member of the matrix-degrading, neutral-metalloproteinase family of enzymes which include collagenase, gelatinase, stromelysin, and telopeptidase. The enzyme may function in the degradation of collagen fibrils by cleaving proteinase-resistant 3/4-collagen fragments that are stabilized by association with neighboring collagen molecules.     

Stimulation of human periodontal ligament fibroblast collagenase production by a gingival epithelial cell-derived factor

To examine whether cell-to-cell interactions between human gingival epithelial cells (HGE) and periodontal ligament fibroblasts (PLF) or gingival fibroblasts (GF) take place in the periodontium, the effects on collagenase production by PLF and GF were analyzed after adding several concentrations of HGE-con-ditioned medium (HGE-CM) to PLF or GF culture. Collagenase production by both cell populations was stimulated by adding HGE-CM, which stimulated collagenase production by PLF to a greater extent than that by GF. The HGE-derived stimulatory factor had a molecular mass of approximately 20 kDa, and its stimulant effect was inhibited markedly in the presence of an anti-human interleukin-lα (IL-lα) neutralizing antibody, indicating that the factor was identical to, or antigenically cross-reactive with, IL-lα. These results suggest that epithelial apical migration in the periodontium may occur after interstitial resident cells have released tissue-degrading enzymes, such as collagenase, and damaged the extracellular matrix, once a sufficient amount of IL-lα-like factor for stimulating the production of proteolytic enzyme has been released by HGE in periodontal lesions.     

Expression of type IV collagen and laminin at the interface between epithelial cells and fibroblasts from human periodontal ligament

The present study was undertaken to examine whether synthesis of type IV collagen and laminin around the epithelial rests of Malassez (ERM) requires direct contact between cells from ERM and periodontal ligament fibroblasts. Human periodontal ligament (HPDL) explants produced outgrowths containing both ERM cells and fibroblasts when cultured in a modified serum-free medium. The interface between ERM cells and fibroblasts was examined using phase-contrast microscopy (PCM) and scanning electron microscopy (SEM). Expression of type IV collagen and laminin was studied by immunohistochemistry and in situ hybridization. It was observed that ERM cells grew underneath fibroblasts or attached to them. At the interface, type IV collagen and laminin and their respective mRNAs were abundant in both ERM cells and fibroblasts, while these proteins and mRNAs showed little if any staining in cells further away from the interface. Hence, these findings indicate that synthesis of type IV collagen and laminin is induced by direct interaction between ERM cells and periodontal ligament fibroblasts.     

Mechanical stress enhances production of cytokines in human periodontal ligament cells induced by Porphyromonas gingivalis

Objective

We have previously reported that human periodontal ligament (hPDL) cells produced many kinds of cytokines as a result of bacterial stimulation, including stimulation with Porphyromonas gingivalis (P. gingivalis). However, the effects of mechanical stress on cytokine production in hPDL cells stimulated by periodontopathogenic bacteria are not clearly understood. In this study, we investigated the effects of mechanical stress on the production of inflammatory cytokines in hPDL cells induced by stimulation with P. gingivalis.

Methods

The hPDL cells were exposed to various levels of mechanical stress (1, 6, 10 and 50 MPa) and costimulated with mechanical stress and P. gingivalis for 24 h. Cytokine mRNA expressions were determined by RT-PCR. Cytokines in the culture supernatant were assessed by ELISA, and morphologic changes in hPDL cells were observed.

Results

The expressions of interleukin (IL)-6, IL-8 and tumor necrosis factor-α mRNA were observed in hPDL cells after exposure to mechanical stress. Moreover, the production of IL-6 and IL-8 increased significantly after exposure to mechanical stress ranging from 1 to 10 MPa. The amount of IL-8 in the culture supernatants of hPDL cells costimulated with P. gingivalis and mechanical stress was significantly higher than the expected additive amount. The morphology of hPDL cells did not change after exposure to 6 MPa, but these cells were partly detached from the Petri dish after exposure to 50 MPa.

Conclusions

These results suggest that local inflammation of the periodontal ligament may be induced mainly by periodontal bacteria, and mechanical stress may promote local inflammation.     

Interleukin-1β stimulates collagenase production by cultured human periodontal ligament fibroblasts

To examine the effects of interleukin-lβ (IL-1β) on collagenase production by human periodontal ligament fibroblasts (PLF) and gingival fibroblasts (GF) in Culture, collagenase activity in conditioned media was determined using a novel procedure that circumvented interference by enzyme inhibitors. Fibroblasts obtained from five paired periodontal ligament and gingival tissues were cultured for two weeks, and then incubated for a further 72 h in α-MEM supplemented with various concentrations of IL-1β (0 to 1250 pg/ml). The conditioned media from individual cultures were harvested and treated with dithiothreitol to inactivate TIMPs, and then with APMA, to activate the latent collagenase. Collagenase activity was measured fluorometrically using FITC-collagen as a substrate. IL-lβ induced a ∼2.4 to 5.2-fold increase in collagenase activity in PLF compared to a ∼1.4 to 2.2-fold increase in GF. These results are in contrast to previous studies in which collagenase activity was measured in the presence of TIMPs, and indicate that PLF are more sensitive to IL-1β than GF. Since both PLF and GF are present in periodontal lesions, it is possible that collagenase secretion stimulated by exposure to inflammatory cell products such as IL-lβ may participate in the destruction of collagen fibers involved in periodontal attachment.     

Human gingival fibroblasts release high‐mobility group box‐1 protein through active and passive pathways

Introduction:  The nuclear protein high‐mobility group box‐1 (HMGB1) acts as a late mediator of inflammation when secreted in the extracellular milieu. In this study, we examined the effect of lipopolysaccharides from periodontal pathogens and apoptotic and necrotic cell death on HMGB1 production in human gingival fibroblasts (HGF). Methods:  HGF from healthy periodontal tissue were cultured and stimulated with lipopolysaccharides (LPS) from Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, and Escherichia coli. We also initiated apoptotic and necrotic cell deaths in HGF. The HMGB1 released in the supernatants from stimulated or dying cells was measured. Immunocytochemical staining against HMGB1 was performed in LPS‐stimulated HGF. Results:  A significantly higher amount of HMGB1 was detected from necrotic and apoptotic HGF. LPS from A. actinomycetemcomitans, P. gingivalis, and E. coli significantly induced the production of HMGB1 in a time‐dependent manner. After 6 h of LPS stimulation, HMGB1 was present in the cytoplasm of cells whereas its location was mainly nuclear after 24 h. Conclusions:  LPS from two major periodontal pathogens, A. actinomycetemcomitans and P. gingivalis, induced HMGB1 secretion from HGF. Apoptotic and necrotic cell deaths resulted in the enhancement of HMGB1. Our results suggest that HGF can be a source of HMGB1 by both active secretion and passive release, and that HMGB1 from HGF may contribute to periodontal tissue destruction.     

Epithelial–mesenchymal interactions induce enamel matrix proteins and proteases in the epithelial cells of the rests of Malassez in vitro

Epithelial–mesenchymal interactions influence morphogenesis and cell differentiation in periodontal tissue regeneration. The current study examined the expression of amelogenin, ameloblastin, matrix metallopeptidase‐20 (MMP‐20), and kallikrein‐4 (KLK‐4) and their effects on the interactions between the epithelial cells of Malassez and periodontal ligament fibroblasts. Explants of human periodontal ligament tissues produced outgrowths containing both the epithelial cells of Malassez and periodontal ligament fibroblasts after incubation in a modified serum‐free medium. Both the epithelial cells and fibroblasts were co‐cultured in the same dish. The distribution and expression of all four factors were evaluated using immunohistochemistry, in‐situ hybridization and RT‐PCR analysis. The epithelial cells of Malassez were cultured separately and were used as the control. Immunohistochemical analysis revealed weak expression of amelogenin, ameloblastin, MMP‐20 and KLK‐4 in epithelial cells of Malassez co‐cultured with periodontal ligament fibroblasts. in‐situ hybridization and RT‐PCR confirmed significant mRNA expression of these factors in co‐cultured cells compared with control cells. MMP20 mRNA was not expressed in control cells. These results suggest that the epithelial–mesenchymal interactions promote differentiation of human epithelial cells of Malassez and that the induction of enamel matrix proteases facilitates the degradation of enamel matrix proteins.     

Toll‐Like Receptor 2 Knockdown Modulates Interleukin (IL)‐6 and IL‐8 but not Stromal Derived Factor‐1 (SDF‐1/CXCL12) in Human Periodontal Ligament and Gingival Fibroblasts

Background: Fibroblasts are now seen as active components of the immune response because these cells express Toll‐like receptors (TLRs), recognize pathogen‐associated molecular patterns, and mediate the production of cytokines and chemokines during inflammation. The innate host response to lipopolysaccharide (LPS) from Porphyromonas gingivalis is unusual inasmuch as different studies have reported that it can be an agonist for Toll‐like receptor 2 (TLR2) and an antagonist or agonist for Toll‐like receptor 4 (TLR4). This study investigates and compares whether signaling through TLR2 or TLR4 could affect the secretion of interleukin (IL)‐6, IL‐8, and stromal derived factor‐1 (SDF‐1/CXCL12) in both human gingival fibroblasts (HGF) and human periodontal ligament fibroblasts (HPDLF). Methods: After small interfering RNA‐mediated silencing of TLR2 and TLR4, HGF and HPDLF from the same donors were stimulated with P. gingivalis LPS or with two synthetic ligands of TLR2, Pam2CSK4 and Pam3CSK4, for 6 hours. IL‐6, IL‐8, and CXCL12 mRNA expression and protein secretion were evaluated by quantitative polymerase chain reaction and enzyme‐linked immunosorbent assay, respectively. Results: TLR2 mRNA expression was upregulated in HGF but not in HPDLF by all the stimuli applied. Knockdown of TLR2 decreased IL‐6 and IL‐8 in response to P. gingivalis LPS, or Pam2CSK4 and Pam3CSK4, in a similar manner in both fibroblasts subpopulations. Conversely, CXCL12 remained unchanged by TLR2 or TLR4 silencing. Conclusion: These results suggest that signaling through TLR2 by gingival and periodontal ligament fibroblasts can control the secretion of IL‐6 and IL‐8, which contribute to periodontal pathogenesis, but do not interfere with CXCL12 levels, an important chemokine in the repair process.     

Collinin Reduces Porphyromonas gingivalis Growth and Collagenase Activity and Inhibits the Lipopolysaccharide‐Induced Macrophage Inflammatory Response and Osteoclast Differentiation and Function

Background: Collinin is a secondary plant metabolite belonging to the class of geranyloxycoumarins. We explored the potential beneficial impact of collinin on periodontal health by investigating its effect on Porphyromonas gingivalis (P. gingivalis), lipopolysaccharide (LPS)‐induced inflammatory response of macrophages, and osteoclastogenesis. Methods: Collinin was synthesized from pyrogallol and propiolic acid. A microdilution assay was used to determine antibacterial activity of collinin. The effect of collinin on collagenase activity of P. gingivalis was determined using fluorescent collagen. Macrophages were treated with collinin before being stimulated with LPS. The secretion of interleukin‐6, chemokine (C‐C motif) ligand 5, and prostaglandin E₂ was assessed by enzyme‐linked immunosorbent assays (ELISA). The inhibitory effect of collinin on differentiation of human preosteoclastic cells was assessed by tartrate‐resistant acid phosphatase staining, whereas the secretion of matrix metalloproteinase‐9 (MMP‐9) was measured by ELISA. Bone resorption activity was investigated by using a human bone plate coupled with an immunoassay that detected the release of collagen fragments. Results: Collinin inhibited the growth of P. gingivalis. The effect was more pronounced under iron‐restricted conditions. Collinin dose dependently inhibited the degradation of type I collagen by P. gingivalis. It was also a potent inhibitor of the LPS‐induced inflammatory response in macrophages and completely inhibited receptor activator of nuclear factor κB ligand‐dependent osteoclast differentiation and MMP‐9 secretion. Last, collinin affected bone degradation mediated by mature osteoclasts by significantly decreasing the release of collagen helical peptides. Conclusion: Although clinical trials are required, data from these in vitro analyses support the potential of collinin as a therapeutic agent for treating inflammatory periodontitis associated with bone breakdown.