Does routine analysis of subgingival microbiota in periodontitis contribute to patient benefit?

In clinical periodontology it is common practice to sample subgingival plaque from periodontitis patients and to search for the presence of alleged periodontal pathogens using routine laboratory techniques such as culture, DNA-DNA hybridization or real-time PCR. Usually, special attention is given to the recognition of 'red complex' microorganisms and to Aggregatibacter actinomycetemcomitans. Recently, molecular open-ended techniques have been introduced which are distinct from the more 'classical' approaches in that they do not preselect for certain species. In this study, we investigated to what extent the outcome of these techniques has changed our insight into the composition of the subgingival microbiota and whether this has consequences on clinical decision making. The open-ended approaches showed that the composition of subgingival plaque is much more complex than previously thought. Next to the 'classical' putative periodontal pathogens, several non-culturable and fastidious species are now recognized as being associated with periodontitis, thus enlarging the group of suspected periodontal pathogens. We conclude that routine analyses of subgingival plaque in the clinic are not necessarily of benefit to the patient.

Alpha11 beta1 integrin-dependent regulation of periodontal ligament function in the erupting mouse incisor.

The fibroblast integrin alpha11beta1 is a key receptor for fibrillar collagens. To study the potential function of alpha11 in vivo, we generated a null allele of the alpha11 gene. Integrin alpha11(-/-) mice are viable and fertile but display dwarfism with increased mortality, most probably due to severely defective incisors. Mutant incisors are characterized by disorganized periodontal ligaments, whereas molar ligaments appear normal. The primary defect in the incisor ligament leads to halted tooth eruption. alpha11beta1-defective embryonic fibroblasts displayed severe defects in vitro, characterized by (i) greatly reduced cell adhesion and spreading on collagen I, (ii) reduced ability to retract collagen lattices, and (iii) reduced cell proliferation. Analysis of matrix metalloproteinase in vitro and in vivo revealed disturbed MMP13 and MMP14 synthesis in alpha11(-/-) cells. We show that alpha11beta1 is the major receptor for collagen I on mouse embryonic fibroblasts and suggest that alpha11beta1 integrin is specifically required on periodontal ligament fibroblasts for cell migration and collagen reorganization to help generate the forces needed for axial tooth movement. Our data show a unique role for alpha11beta1 integrin during tooth eruption.

Transmission Electron Microscopy of Bone.

Electron microscopic analysis of mineralized tissues like bone and dentin is essential for understanding of cell-cell/cell-matrix interactions, and the three-dimensional organization of these tissues. This chapter describes a few methods to process mineralized tissues obtained from different sources for ultrastructural analysis by transmission electron microscopy.

Plasminogen activators are involved in the degradation of bone by osteoclasts.

Osteoclastic bone degradation depends on the activity of several proteolytic enzymes, in particular to those belonging to the classes of cysteine proteinases and matrix metalloproteinases (MMPs). Yet, several findings suggest that the two types of plasminogen activators (PA), the tissue- and urokinase-type PA (tPA and uPA, respectively) are also involved in this process. To investigate the involvement of these enzymes in osteoclast-mediated bone matrix digestion, we analyzed bone explants of mice that were deficient for both tPA and uPA and compared them to wild type mice. The number of osteoclasts as well as their ultrastructural appearance was similar for both genotypes. Next, calvarial and metatarsal bone explants were cultured for 6 or 24 h in the presence of selective inhibitors of cysteine proteinases or MMPs and the effect on osteoclast-mediated bone matrix degradation was assessed. Inhibition of the activity of cysteine proteinases in explants of control mice resulted in massive areas of non-digested demineralized bone matrix adjacent to the ruffled border of osteoclasts, an effect already maximal after 6 h. However, at that time point these demineralized areas were not observed in bone explants from uPA/tPA deficient mice. After prolonged culturing (24 h), a comparable amount of demineralized bone matrix adjacent to actively resorbing osteoclasts was observed in the two genotypes, suggesting that degradation was delayed in uPA/tPA deficient bones. The activity of cysteine proteinases as assessed in bone extracts, proved to be higher in extracts from uPA/tPA(-/-) bones. Immunolocalization of the integrin alpha(v)beta(3) of in vitro generated osteoclasts demonstrated a more diffuse labeling of osteoclasts derived from uPA/tPA(-/-) mice. Taken together, our data indicate that the PAs play a hitherto unrecognized role in osteoclast-mediated bone digestion. The present findings suggest that the PAs are involved in the initial steps of bone degradation, probably by a proper integrin-dependent attachment to bone.

Osteoclastic bone degradation and the role of different cysteine proteinases and matrix metalloproteinases: differences between calvaria and long bone.

UNLABELLED: Osteoclastic bone degradation involves the activity of cathepsin K. We found that in addition to this enzyme other, yet unknown, cysteine proteinases participate in digestion. The results support the notion that osteoclasts from different bone sites use different enzymes to degrade the collagenous bone matrix. INTRODUCTION: The osteoclast resorbs bone by lowering the pH in the resorption lacuna, which is followed by secretion of proteolytic enzymes. One of the enzymes taken to be essential in resorption is the cysteine proteinase, cathepsin K. Some immunolabeling and enzyme inhibitor data, however, suggest that other cysteine proteinases and/or proteolytic enzymes belonging to the group of matrix metalloproteinases (MMPs) may participate in the degradation. In this study, we investigated whether, in addition to cathepsin K, other enzymes participate in osteoclastic bone degradation. MATERIALS AND METHODS: In bones obtained from mice deficient for cathepsin K, B, or L or a combination of K and L, the bone-resorbing activity of osteoclasts was analyzed at the electron microscopic level. In addition, bone explants were cultured in the presence of different selective cysteine proteinase inhibitors and an MMP inhibitor, and the effect on resorption was assessed. Because previous studies showed differences in resorption by calvarial osteoclasts compared with those present in long bones, in all experiments, the two types of bone were compared. Finally, bone extracts were analyzed for the level of activity of cysteine proteinases and the effect of inhibitors hereupon. RESULTS: The analyses of the cathepsin-deficient bone explants showed that, in addition to cathepsin K, calvarial osteoclasts use other cysteine proteinases to degrade bone matrix. It was also shown that, in the absence of cathepsin K, long bone osteoclasts use MMPs for resorption. Cathepsin L proved to be involved in the MMP-mediated resorption of bone by calvarial osteoclasts; in the absence of this cathepsin, calvarial osteoclasts do not use MMPs for resorption. Selective inhibitors of cathepsin K and other cysteine proteinases showed a stronger effect on calvarial resorption than on long bone resorption. CONCLUSIONS: Our findings suggest that (1) cathepsin K-deficient long bone osteoclasts compensate the lack of this enzyme by using MMPs in the resorption of bone matrix; (2) cathepsin L is involved in MMP-mediated resorption by calvarial osteoclasts; (3) in addition to cathepsin K, other, yet unknown, cysteine proteinases are likely to participate in skull bone degradation; and finally, (4) the data provide strong additional support for the existence of functionally different bone-site specific osteoclasts.

Loss-of-function mutations in cathepsin C in two families with Papillon-Lefèvre syndrome are associated with deficiency of serine proteinases in PMNs.

Papillon-Lefèvre syndrome (PLS) is a rare autosomal recessive disease that involves severe periodontitis and hyperkeratosis of the hand palms and foot soles. Recently it was found that PLS patients carry loss-of-function mutations in the gene encoding cathepsin C (CTSC). In the present study we have analyzed the CTSC gene in two unrelated families with PLS. In the first non-consanguineous family, mutation analysis revealed the previously reported c.815G>C/p.R272P mutation. The second consanguineous family displayed a c.1213C>A mutation which resulted in the novel mutation p.H405N and is the first mutation described in the active site of the enzyme. The PLS patients had, next to the absence of cathepsin C activity in polymorphonuclear leukocytes (PMNs), no activity of the three serine proteinases elastase, cathepsin G and proteinase 3. Serine proteinases are supposed to be important in both the innate and adaptive immune systems. Their absence in PLS patients could explain the inadequate defense to periodontal infection.

Collagen type I, III and V differently modulate synthesis and activation of matrix metalloproteinases by cultured rabbit periosteal fibroblasts.

In the present study we investigated whether the collagen types I, III and V affect the activity of fibroblasts obtained from rabbit periosteum. The cells were cultured on plates either or not coated with different amounts of collagen type I, III or V and analyzed for their attachment, DNA synthesis and the expression and activity of matrix metalloproteinases (MMPs). Our data show that the three collagen types promoted attachment and spreading of the cells and stimulated DNA synthesis when used in relatively low concentrations. High concentrations of type V-but not of type I or III-proved to inhibit thymidine incorporation. The expression and activity of matrix metalloproteinase 1 (MMP-1; interstitial collagenase) decreased under the influence of relatively low amounts of collagen (<40 microg/well), whereas higher levels increased its release. Matrix metalloproteinase 2 (MMP-2; gelatinase A) was up-regulated by the different types of collagen; the active fraction of stromelysin-1 (MMP-3) decreased. Accordingly, the mRNA expression of MMP-1 and -3 were reduced. The expression of MMP-2 mRNA, however, proved to be unaffected. Blocking antibodies to beta(1)-integrin or echistatin increased the level of MMP-1 but had no effect on MMP-2. All parameters tested were similarly affected by type I and III collagen, whereas the effect of type V was always less. We conclude that the collagen types I, III and V provide different sets of signals for fibroblasts that differently modulate their proliferation and MMP expression.

Site-specific localization of two distinct phosphatases along the osteoblast plasma membrane: tissue non-specific alkaline phosphatase and plasma membrane calcium ATPase.

In osteoblasts, alkaline phosphatase has been reported to be restricted to the basolateral domains. In recent studies, we have demonstrated phosphatase activities different from those of tissue non-specific alkaline phosphatase (TNSALP) along the osteoidal aspect of osteoblast membrane at alkaline and neutral pH on undecalcified freshly frozen sections of rat bones. In the present study, we sought to further characterize and define the nature of membrane-associated phosphatases along the osteoidal aspect of osteoblasts. Histochemical properties of the enzymes and their localization in vivo were examined in long bones of normal Wistar rats and TNSALP null mutant mice and their wild type littermates. Molecular profiles of the enzymes in the osteoblast extracts were also examined. The enzymatic activity of the phosphatase along the osteoidal surface of osteoblasts proved to be activated by both Mg2+ and Ca2+. Unlike TNSALP, the activity was inhibited by vanadate but resistant to levamisole, implicating a similarity between this enzyme and plasma membrane Ca2+ transport ATPase (PMCA). Immunohistochemistry showed that PMCA immunoreactions were restricted to the osteoidal domain of the plasma membrane. Native-PAGE analysis of osteoblast extracts suggested the presence of two phosphatases corresponding, respectively, to TNSALP and PMCA. Western blot analysis after SDS-PAGE of osteoblast extracts confirmed the existence of PMCA (140 kDa) and TNSALP (80 kDa). Gel-chemical analysis of the osteoblast extract from TNSALP null mutant mice depicted phosphatase activity, which was resistant to levamisole. These data suggest the presence of a phosphatase different from TNSALP, most plausibly PMCA, on the osteoidal surface of osteoblasts.

Effect of an oxygenating agent on oral bacteria in vitro and on dental plaque composition in healthy young adults.

UNLABELLED: Oral bacteria live in symbiosis with the host. Therefore, when mouthwashes are indicated, selective inhibition of taxa contributing to disease is preferred instead of broad-spectrum antimicrobials. The potential selectivity of an oxygenating mouthwash, Ardox-X® (AX), has not been assessed. The aim of this study was to determine the antimicrobial potential of AX and the effects of a twice-daily oral rinse on dental plaque composition. MATERIAL AND METHODS: In vitro, 16 oral bacterial strains were tested using agar diffusion susceptibility, minimum inhibitory and minimum bactericidal concentration tests. A pilot clinical study was performed with 25 healthy volunteers. Clinical assessments and microbiological sampling of supragingival plaque were performed at 1 month before the experiment (Pre-exp), at the start of the experiment (Baseline) and after the one-week experimental period (Post-exp). During the experiment individuals used AX mouthwash twice daily in absence of other oral hygiene measures. The microbiological composition of plaque was assessed by 16S rRNA gene amplicon sequencing. RESULTS: AX showed high inter-species variation in microbial growth inhibition. The tested Prevotella strains and Fusobacterium nucleatum showed the highest sensitivity, while streptococci and Lactobacillus acidophilus were most resistant to AX. Plaque scores at Pre-exp and Baseline visits did not differ significantly (p = 0.193), nor did the microbial composition of plaque. During a period of 7-days non-brushing but twice daily rinsing plaque scores increased from 2.21 (0.31) at Baseline to 2.43 (0.39) Post-exp. A significant microbial shift in composition was observed: genus Streptococcus and Veillonella increased while Corynebacterium, Haemophilus, Leptotrichia, Cardiobacterium and Capnocytophaga decreased (p ≤ 0.001). CONCLUSION: AX has the potential for selective inhibition of oral bacteria. The shift in oral microbiome after 1 week of rinsing deserves further research.

Transmission electron microscopy of bone.

This chapter describes procedures to process mineralized tissues obtained from different sources for transmission electron microscopy (TEM). Methods for fixation, resin embedding, staining of semi-thin sections and ultrathin sections are presented. In addition, attention will be paid to processing of cultured bone explants for TEM analysis.

LAMP-2: a control step for phagosome and autophagosome maturation.

The two structurally related, major lysosomal membrane proteins LAMP-1 and LAMP-2 were for a long time regarded as crucial for the protection of the lysosomal membrane from the hostile lumenal environment. However, recent studies on the effects of single and combined LAMP-deficiency in mice reveal alternative functions. LAMP proteins, but especially LAMP-2, are important regulators in successful maturation of both autophagosomes and phagosomes. LAMP-2 deficiency causes an accumulation of autophagosomes in many tissues leading to cardiomyopathy and myopathy in mice and patients suffering from Danon Disease. The central role of LAMP-2 is also underlined by a recent study where LAMP-2 knockout mice are shown to have an impaired phagosomal maturation in neutrophils. The impairment of this important innate immune defense process in these mice leads to periodontitis, one of the most widespread infectious diseases worldwide. The retarded clearance of bacterial pathogens was probably due to an inefficient fusion capacity between lysosomes and phagosomes. Recent studies in LAMP double-knockout fibroblasts suggests that LAMP-deficiency impairs the dynein-mediated transport of lysosomes to perinuclear regions where fusion with (auto)phagosomes occurs.

Impaired phagosomal maturation in neutrophils leads to periodontitis in lysosomal-associated membrane protein-2 knockout mice.

Inflammatory periodontal diseases constitute one of the most common infections in humans, resulting in the destruction of the supporting structures of the dentition. Circulating neutrophils are an essential component of the human innate immune system. We observed that mice deficient for the major lysosomal-associated membrane protein-2 (LAMP-2) developed severe periodontitis early in life. This development was accompanied by a massive accumulation of bacterial plaque along the tooth surfaces, gingival inflammation, alveolar bone resorption, loss of connective tissue fiber attachment, apical migration of junctional epithelium, and pathological movement of the molars. The inflammatory lesions were dominated by polymorphonuclear leukocytes (PMNs) apparently being unable to efficiently clear bacterial pathogens. Systemic treatment of LAMP-2-deficient mice with antibiotics prevented the periodontal pathology. Isolated PMNs from LAMP-2-deficient mice showed an accumulation of autophagic vacuoles and a reduced bacterial killing capacity. Oxidative burst response was not altered in these cells. Latex bead and bacterial feeding experiments showed a reduced ability of the phagosomes to acquire an acidic pH and late endocytic markers, suggesting an impaired fusion of late endosomes-lysosomes with phagosomes. This study underlines the importance of LAMP-2 for the maturation of phagosomes in PMNs. It also underscores the requirement of lysosomal fusion events to provide sufficient antimicrobial activity in PMNs, which is needed to prevent periodontal disease.

Structure of the periodontium in cathepsin C-deficient mice.

Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency leads to changes in the structure of the periodontal tissues as a result of which its barrier function to pathogens is compromised. We studied the structure of the periodontium in 9-month-old cathepsin C-deficient mice (cathepsin C(-/-)) and compared this with age-matched wild-type mice. Our observations showed that the overall structure of the gingiva, periodontal ligament, alveolar process, and cementum layer are normal in cathepsin C(-/-) mice, with one exception, namely that epithelial rests of Malassez in the periodontal ligament of the cathepsin C(-/-) mice are slightly enlarged. In both experimental and control animals, we noted cyst formation in rests of Malassez. No signs of periodontal infection were observed. It is concluded that cathepsin C deficiency does not lead to major changes in the structure of the periodontium.

Gingival fibroblasts are better at inhibiting osteoclast formation than periodontal ligament fibroblasts.

Various studies indicate that periodontal ligament fibroblasts (PLF) have some similarities to osteoblasts, for example they have the capacity to induce the formation of osteoclast-like cells. Here, we investigated whether a second population of tooth-associated fibroblasts, gingival fibroblasts (GF), has similar osteoclastogenesis properties. PLF and GF were co-cultured with peripheral blood mononuclear cells (PBMC) in the presence and absence of dexamethasone and 1alpha,25dihydroxycholecalciferol (dex + vit D(3)) on plastic and on cortical bone slices. Tartrate resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs) were more abundant in co-cultures with PLF than in GF-PBMC co-cultures, more abundant on plastic compared to bone and more abundant in the presence of dex + vit D(3). In line with these findings was an inhibition of MNC formation and not inhibition of existing osteoclasts by medium conditioned by GF. We next investigated whether expression of molecules important for osteoclastogenesis differed between the two types of fibroblasts and whether these molecules were regulated by dex + vit D(3). OPG was detected at high levels in both fibroblast cultures, whereas RANKL could not be detected. Resorption of bone did not occur by the MNCs formed in the presence of either fibroblast subpopulation, suggesting that the fibroblasts secrete inhibitors of bone resorption or that the osteoclast-like cells were not functional. The incapacity of the MNCs to resorb was abolished by culturing the fibroblast-PBMC cultures with M-CSF and RANKL. Our results suggest that tooth-associated fibroblasts may trigger the formation of osteoclast-like cells, but more importantly, they play a role in preventing bone resorption, since additional stimuli are required for the formation of active osteoclasts.

Role of polymorphonuclear leukocyte-derived serine proteinases in defense against Actinobacillus actinomycetemcomitans.

Periodontitis is a chronic destructive infection of the tooth-supportive tissues, which is caused by pathogenic bacteria such as Actinobacillus actinomycetemcomitans. A severe form of periodontitis is found in Papillon-Lefèvre syndrome (PLS), an inheritable disease caused by loss-of-function mutations in the cathepsin C gene. Recently, we demonstrated that these patients lack the activity of the polymorphonuclear leukocyte (PMN)-derived serine proteinases elastase, cathepsin G, and proteinase 3. In the present study we identified possible pathways along which serine proteinases may be involved in the defense against A. actinomycetemcomitans. Serine proteinases are capable to convert the PMN-derived hCAP-18 into LL-37, an antimicrobial peptide with activity against A. actinomycetemcomitans. We found that the PMNs of PLS patients released lower levels of LL-37. Furthermore, because of their deficiency in serine proteases, the PMNs of PLS patients were incapable of neutralizing the leukotoxin produced by this pathogen, which resulted in increased cell damage. Finally, the capacity of PMNs from PLS patients to kill A. actinomycetemcomitans in an anaerobic environment, such as that found in the periodontal pocket, seemed to be reduced. Our report demonstrates a mechanism that suggests a direct link between an inheritable defect in PMN functioning and difficulty in coping with a periodontitis-associated pathogen.

Effect of CD44 deficiency on in vitro and in vivo osteoclast formation.

In vitro studies have shown that CD44 is involved in the fusion process of osteoclast precursor cells. Yet, in vivo studies do not support this, since an osteopetrotic phenotype has not been described for CD44 knock-out (CD44 k.o.) mice. This discrepancy may suggest that the role of CD44 in fusion may depend on the microenvironment of osteoclast formation. We investigated osteoclast formation of CD44 k.o. and wild-type mice under three conditions: in vitro, both on plastic and on bone and in vivo by analyzing osteoclast number, and size in long bones from wild-type and CD44 k.o. mice. Bone marrow cells from wild-type and CD44 k.o. mice were analyzed for their capacity to form osteoclasts on plastic and on bone in the presence of macrophage colony stimulating factor (M-CSF) and receptor activator of NF-kB ligand (RANKL). On plastic, the number of multinucleated tartrate resistant acid phosphatase (TRAP) positive cells in CD44 k.o. cultures was twofold higher than in wild-type cultures. On bone, however, equal numbers of osteoclasts were formed. Interestingly, the total number of osteoclasts formed on bone proved to be higher than on plastic for both genotypes, strongly suggesting that osteoclastogenesis was stimulated by the bone surface, and that CD44 is not required for osteoclast formation on bone. Functional analyses showed that bone resorption was similar for both genotypes. We further studied the osteoclastogenic potential of wild-type bone marrow cells in the presence of CD44 blocking antibodies. Osteoclastogenesis was not affected by these antibodies, a further indication that CD44 is not required for the formation of multinucleated cells. Finally, we analyzed the in vivo formation of osteoclasts by analyzing long bones from wild-type and CD44 k.o. mice. Morphometric analysis revealed no difference in osteoclast number, nor in number of nuclei per osteoclasts or in osteoclast size. Our in vitro experiments on plastic showed an enhanced formation of osteoclasts in the absence of CD44, thus suggesting that CD44 has an inhibitory effect on osteoclastogenesis. However, when osteoclasts were generated on bone, no differences in number of multinucleated cells nor in bone resorption were seen. These observations are in agreement with in vivo osteoclast characteristics, where no differences between wild-type and CD44 k.o. bones were encountered. Therefore, the modulating role of CD44 in osteoclast formation appears to depend on the microenvironment.

Involvement of matrix metalloproteinases in the onset of dentin mineralization.

In order to study the involvement of matrix metalloproteinases (MMPs) on dentin formation and mineralization, day 18 embryonic mouse tooth germs were cultured for 10 d in the presence or absence of Marimastat, a general MMP inhibitor, or CT(1166), a more selective inhibitor of gelatinases (MMP-2 and MMP-9) and stromelysin-1 (MMP-3). With Marimastat a dose-dependent increase in thickness of the predentin layer and a decreased mineralization of dentin were observed. At the highest concentration of the inhibitor used, enamel formation had ceased. With CT(1166), these effects were already apparent at the lowest concentration used. Western blot analyses demonstrated that the two inhibitors inhibited the expression of enamelysin (MMP-20). These observations indicate that MMPs (possibly MMP-2, -3, -9 and/or -20) play a role in the onset of dentin mineralization. The lack of enamel formation was possibly due to diffusion of amelogenin from its normal site of apposition. The protein clearly was not retained at the surface of the non-mineralized dentin layer, and immunopositive amelogenin accumulated in the odontoblast compartment. The diffusion of enamel proteins and the accumulation revealed by immunolabeling of two small leucine-rich proteoglycans, decorin and biglycan, in the predentin may have contributed to impaired dentin mineralization.

On the role of MT1-MMP, a matrix metalloproteinase essential to collagen remodeling, in murine molar eruption and root growth.

Although the connective tissues of the periodontium are subject to a high turnover rate, no conclusive evidence has yet emerged that periodontal collagen turnover is essential for the eruption of teeth or for root elongation. These processes were studied in mice deficient in MT1-MMP, a membrane type matrix metalloproteinase essential for remodeling of soft tissue-hard tissue interfaces. Mandibular first molars of deficient mice and their wild-type littermates were subjected to stereological analysis in order to assess root length, eruption and the volume density of phagocytosed collagen in periodontal ligament fibroblasts. The data showed that both eruption and root elongation were severely inhibited in animals lacking the enzyme. We also found, in periodontal ligament fibroblasts from MT1-MMP-deficient mice, a massive age-related accumulation (up to 60-fold over controls) of collagen fibril-containing phagosomes. Phagolysosomes, which represent the next downstream step in collagen fibril degradation by the lysosomal pathway, did not accumulate. These observations indicate that MT1-MMP plays a central role in periodontal remodeling. The stunted root growth and the failure to erupt indicate the important role of the enzyme in tooth development.