Immunohistochemical localisation of extracellular matrix proteins in the periodontium during cementogenesis in the rat molar

OBJECTIVE: The development of the periodontium involves the coordinated expression of numerous extracellular matrix (ECM) macromolecules and their receptors (integrins). The aim of this study was to determine the expression of selected hard and soft tissue matrix molecules and the integrin alpha5beta1 in the periodontal tissues, during cementogenesis in the rat molar. METHODS: Using immunohistochemical methods, the distribution of the extracellular matrix proteins, fibronectin, tenascin, and bone sialoprotein (BSP), as well as the integrin subunits alpha5 and beta1 were studied in rats aged 3, 5 and 8 weeks. RESULTS: Fibronectin was widely distributed in the gingival epithelium, gingival connective tissue and in the periodontal ligament. Tenascin expression was less marked compared with fibronectin, but was more distinctly associated with cells and peri-cellular areas of the epithelial-connective tissue interface, the gingiva and within the periodontal ligament. The fibronectin-receptor alpha5beta1 integrins were expressed by epithelial cells, periodontal ligament cells and gingival fibroblasts. A notable finding was the increased staining intensity of fibronectin, tenascin and alpha5beta1 integrin in all 5-week old molar sections in the periodontal ligament matrix and cells, apical to the cemento-enamel junction (CEJ) along the alveolar crest (AC) ridge height. Bone sialoprotein was distinctly associated with the hard tissues of the periodontium as acellular cementum and alveolar bone matrix expressed bone sialoprotein throughout all sections, in all age groups. CONCLUSIONS: In conclusion, this study has demonstrated the selective distribution of several hard and soft tissue matrix molecules during periodontogenesis. The results highlight the complex nature of interactions of various proteins and molecules during development. The interactions between these molecules and their specific roles in development and regeneration await further investigation.     

Autocrine growth factors in human periodontal ligament cells cultured on enamel matrix derivative

OBJECTIVE: Enamel extracellular matrix proteins in the form of the enamel matrix derivative EMDOGAIN (EMD) have been successfully employed to mimic natural cementogenesis to restore fully functional periodontal ligament, cementum and alveolar bone in patients with severe periodontitis. When applied to denuded root surfaces EMD forms a matrix that locally facilitates regenerative responses in the adjacent periodontal tissues. The cellular mechanism(s), e.g. autocrine growth factors, extracellular matrix synthesis and cell growth, underlying PDL regeneration with EMD is however poorly investigated. MATERIAL AND METHODS: Human periodontal ligament (PDL) cells were cultured on EMD and monitored for cellular attachment rate, proliferation, DNA replication and metabolism. Furthermore, intracellular cyclic-AMP levels and autocrine production of selected growth factors were monitored by immunological assays. Controls included PDL and epithelial cells in parallel cultures. RESULTS: PDL cell attachment rate, growth and metabolism were all significantly increased when EMD was present in cultures. Also, cells exposed to EMD showed increased intracellular cAMP signalling and autocrine production of TGF-beta1, IL-6 and PDGF AB when compared to controls. Epithelial cells increased cAMP and PDGF AB secretion when EMD was present, but proliferation and growth were inhibited. CONCLUSION: Cultured PDL cells exposed to EMD increase attachment rate, growth rate and metabolism, and subsequently release several growth factors into the medium. The cellular interaction with EMD generates an intracellular cAMP signal, after which cells secrete TGF-beta1, IL-6 and PDGF AB. Epithelial cell growth however, is inhibited by the same signal. This suggest that EMD favours mesenchymal cell growth over epithelium, and that autocrine growth factors released by PDL cells exposed to EMD contribute to periodontal healing and regeneration in a process mimicking natural root development.     

Effect of methyl mercaptan on synthesis and degradation of collagen

Measurements of the conversion of [¹⁴C]-proline to [¹⁴C]-hydroxyproline were employed to assess the effect of methyl mercaptan on intra- and extracellular metabolism of collagenous proteins in human gingival fibroblast cultures. Following a 30-min pulse, 10 ng of methyl mercaptan per ml of 95% air/5% CO₂, headspace suppressed collagen synthesis by 39% and increased the intracellular degradation of newly synthesized collagen from 26% to 42%. Parallel cultures assayed for proline transport demonstrated a 29% inhibition of [¹⁴C]-proline uptake. A similar analysis of cultures exposed to methyl mercaptan for 12 h revealed an increase in intracellular degradation (20% control vs. 30% test) and a marked increase in extracellular collagenolysis (4% control vs. 55% test). While pulsing, collagen synthesis was decreased by 39%. Slab gel electrophoresis also demonstrated that treatment with methyl mercaptan caused reductions both in mature a, and α₂ chains of type I collagen and in type III procollagen. Identities of the procollagen species were confirmed by pepsin digestion. Reverse transcribed polymerase chain reaction was utilized to compare expression of a1 chains of type I procollagen with type III procollagen and indicated suppression of mRNA synthesis for type III procollagen in cultures exposed to methyl mercaptan.     

Specific pro-apoptotic fibronectin fragments modulate proteinase expression in periodontal ligament cells

BACKGROUND: During inflammation and periodontal disease, the extra-cellular matrix protein fibronectin (FN) is degraded into fragments whose activities differ from those of the intact molecule. In periodontal ligament cells in vitro, some fragments elevate the expression of matrix metalloproteinases and serine proteinases, while others induce apoptosis through a mechanism regulated by caspase proteases and specific signaling pathways. In this study, we investigated whether the pro-apoptotic V+H- fibronectin fragment modulates proteinase expression as part of the apoptotic mechanism in periodontal ligament cells. The importance of this study is that a structurally similar fibronectin fragment found in vivo (40-kDa) is associated with periodontal disease status. However, the recombinant V+H- FN protein which is structurally and functionally similar to the in vivo 40-kDa fragment was chosen as the focus of these studies over the in vivo fragment because of the ability to readily produce and purify large quantities of recombinant fragments for detailed studies. Thus, findings from this study relate to our understanding of how the extracellular matrix of the periodontal ligament in an inflamed environment may contribute to periodontal disease and its progression. METHODS: We used substrate zymography, reverse zymography, proteinase inhibitors, and partial sequencing to investigate whether the pro-apoptotic V+H- fibronectin fragment modulates proteinase expression as part of this apoptotic mechanism. RESULTS: Incubation with the V+H- fragment reduced the expression of all gelatinolytic proteinases and inhibitors commonly expressed by periodontal ligament cells. In the presence of caspase inhibitors, inhibitors known to suppress apoptosis, however, the reduced proteinase profile was rescued, showing that caspase inhibitors were able to reverse the reduced proteinase profile and indicating that caspase-mediated pathways are pertinent to fibronectin-fragment-mediated matrix metalloproteinase expression. In addition, the V+H- fragment also triggered the expression of a unique high molecular weight gelatinolytic proteinase of approximately 200 kDa. This proteinase was a serine proteinase, whose identity is not known. CONCLUSION: These findings suggest that matrix-degrading proteinases may be involved in apoptosis as part of a unique mechanism of periodontal tissue breakdown, in which novel proteinases may help execute the dissolution of the extracellular matrix.     

Effects of transforming growth factor-beta 1 on cultured dental follicle cells from rat mandibular molars.

Analysis of the total proteins secreted by cultured dental follicle cells revealed that transforming growth factor-beta 1 (TGF-beta 1) stimulated them to secrete more extracellular matrix proteins into a serum-free medium than did follicle cells not exposed to the growth factor. Electrophoresis and scanning densitometry showed that secretion of all the major proteins was increased by exposure to the growth factor but the amounts ranged from a 66% increase for one of the procollagen chains to a 7% increase for fibronectin. Immunofluorescence using anti-type I collagen and anti-fibronectin showed that the intracellular concentration and intracellular localization of the antibodies was not changed by incubating the cells with the growth factor. The growth factor did not cause an increase in cell number but did modify the association of the cells in the culture, causing them to aggregate into clusters whereas the control cells formed a confluent monolayer. These results suggest that TGF-beta 1 may signal the fibroblasts of the dental follicle to secrete the extracellular matrix needed for its development into a periodontal ligament.     

Epithelial cell-fibroblast interactions: modulation of extracellular matrix proteins in cultured oral cells

A model system involving co-cultures of human gingival or periodontal ligament fibroblasts with mouse epithelial root sheath cells or human gingival eipthelial cells was used to study epithelial cell-fibroblast interactions. Double-labeled immunofluorescence and microfluorometry were used to investigate the expression of extracellular martix molecules of collagen type I (collagen I), type III (collagen III) and fibronectin in fibroblasts. When fibroblasts from either source were cultured alone, the fluorescence for collagen I and fibronectin ranged from strongly positive to almost negative. Collagen III staining was relatively weak compared with that of collagen I. After 2-3 days of co-culture, gingival fibroblasts and ligament fibroblasts adjacent to the mouse sheath cells exhibitied enhanced intracellular fluorescence for collagen I and fibronectin. Very little change was observed for collagen III. Gingival fibroblasts cultured with gingival epithelial cells showed increased fluorescence for collagen I but decreased fluorescence for fibronectin. In contrast, the fluorescence intensity for both collagen I and fibronectin in ligament fibroblasts were reduced after 3 days of co-culture with gingival epithelial cells. Ultrastructural changes in fibroblasts co-cultured with mouse root sheath cells included increased Golgi cisternae and vesicles and an increased abundance of rough endoplasmic reticulum, polyribosomes, secretory vesicles and pinocytotic vesicles. Thus, the expression of extracellular martix proteins and the metabolic activity of fibroblasts can be modulated by oral epithelial cells.     

Expression of fibronectin and integrins in cultured periodontal ligament epithelial cells.

The process of attachment of epithelial cells obtained from the porcine periodontal ligament (cell rests of Malassez) to different extracellular matrix proteins and their expression of fibronectin and integrin receptors were studied by means of immunocytochemistry, in situ hybridization, and time-lapse cinemicrography techniques. The cell lines of periodontal ligament epithelial cells (PLE cells) attached to and spread rapidly on fibronectin, vitronectin, and type I collagen. One of the cell lines also attached to laminin, while the other cell line showed poor attachment to both laminin and Matrigel, a basement membrane material. By use of the in situ hybridization technique, some PLE cells were found to express the fibronectin gene strongly. Immunocytochemical staining localized fibronectin in extracellular fibrils and intracellular granules. Fibronectin was also found in the tracks left behind by the cells migrating on the substratum. Arg-gly-asp-ser peptide inhibited the attachment of the PLE cells to fibronectin, laminin, type I collagen, and vitronectin by 47%, 43%, 83%, and 94%, respectively, suggesting that the cell-matrix interactions were partly mediated by receptors related to the integrin family. Antibodies against the beta 1-integrin subunit stained the cell bodies and the plasma membrane projections of spreading cells. After 24 h or longer in culture, beta 1-integrins were localized to the regions of cell-cell contact. Cinemicrography of the arg-gly-asp-ser-peptide-treated cells demonstrated that the spreading and migration of isolated cells were prevented by the peptide. The peptide did not appear to dissociate the cell-cell contacts or interfere with migration of spread-cell colonies.(ABSTRACT TRUNCATED AT 250 WORDS)     

The relationship between oral malodor, gingivitis, and periodontitis. A review.

Volatile sulfur compounds (VSC) are a family of gases which are primarily responsible for halitosis, a condition in which objectionable odors are present in mouth air. Although most patients perceive this condition as primarily a cosmetic problem, an increasing volume of evidence is demonstrating that extremely low concentrations of many of these compounds are highly toxic to tissues. VSC may, therefore, play a role in the pathogenesis of inflammatory conditions such as periodontitis. Since these compounds result from bacterial putrefaction of protein, investigations have been conducted to determine whether specific bacteria are associated with odor production. Two members of this family, hydrogen sulfide (H2S) and methyl mercaptan (CH3SH), are primarily responsible for mouth odor. Although many bacteria produce H2S, the production of CH3SH, especially at high levels, is primarily restricted to periodontal pathogens. Direct exposure to either of these metabolites adversely affects protein synthesis by human gingival fibroblasts in culture. However, methyl mercaptan has the greatest effect. Other in vitro experiments have demonstrated that cells exposed to methyl mercaptan synthesize less collagen, degrade more collagen, and accumulate collagen precursors which are poorly cross-linked and susceptible to proteolysis. CH3SH also increases permeability of intact mucosa and stimulates production of cytokines which have been associated with periodontal disease. VSC, and in particular methyl mercaptan, are therefore capable of inducing deleterious changes in both the extracellular matrix and the local immune response of periodontal tissues to plaque antigens. This article reviews these data and emphasizes the potential importance of VSC in the transition of periodontal tissues from clinical health to gingivitis and then to periodontitis.     

Enamel matrix proteins bind to wound matrix proteins and regulate their cell-adhesive properties

Enamel matrix proteins (EMP) induce periodontal regeneration and accelerate dermal wound healing, but the cellular mechanisms of these processes are unclear. We investigated the binding of EMP to the wound matrix proteins fibronectin, laminin-1, collagen type I, and collagen type IV and analyzed the interaction of epithelial cells and periodontal ligament fibroblasts (PDLF) with EMP and composite matrices of EMP + fibronectin or EMP + collagen. The adhesion of PDLF to EMP was concentration- and integrin-dependent and did not require de novo protein synthesis. EMP supported PDLF migration. In contrast, keratinocytes did not adhere to EMP if their protein synthesis was blocked. EMP showed concentration-dependent binding of fibronectin, peaking at 100 microg ml(-1) (before the precipitation point) of EMP. Type I collagen binding to EMP peaked at a low (1 microg ml(-1)) and narrow concentration range. Neither laminin-1 nor type IV collagen bound to EMP. Collagen and fibronectin, bound to EMP, showed significantly reduced (> 50%) binding of both epithelial cells and PDLF compared with the equivalent concentration of these proteins alone. PDLF, but not epithelial cell, adhesion was rescued by increasing the EMP concentration. These findings show that EMP binds to wound extracellular matrix proteins and regulates their adhesive properties. Such interactions may favor fibroblast adhesion over epithelial cells, potentially promoting connective tissue regeneration.     

Immunoelectron microscopic studies on the distributions of fibronectin and actin in a cellular dense connective tissue: the periodontal ligament of the rat

Mouse monoclonal anti-fibronectin antibodies and rabbit affinity-purified actin antibodies were used with gold-labelled secondary antibodies to study the distributions of fibronectin and actin within the periodontal tissues of the rat. Following perfusion fixation, demineralization, and embedding in glycolmethacrylate, sections 70–80 nm thick were obtained from the gingiva, periodontal ligament and the supporting bone of the first and second mandibular molars. In both gingiva and periodontal ligament, fibronectin was located over the collagen fibers and at certain sites at the cell-collagen interface. In addition, unbanded fibrillar material 10–20 nm thick was decorated by fibronectin antibodies. Actin was localized primarily in the peripheral cytoplasm and at the cell-extracellular matrix interface. Labelling for actin was detected at a few sites within the extracellular matrix. In double labelling experiments, fibronectin and actin were observed to co-distribute at cell-matrix interfaces. These data support the concept that fibronectin within the dense connective tissue of the rat gingiva and periodontal ligament, as in other soft connective tissues, may mediate the interaction between different components of the extracellular matrix and between these components and the cell membrane. In addition, they strengthen the hypothesis that actin filaments may be active in vivo in the formation of cell-matrix contacts, and that actin and fibronectin may form fibronexus-like structures similar to those described for fibroblasts in culture.     

Molecules Contributing to the Maintenance of Periodontal Tissues

This review is aimed at providing a depiction of molecules and their topography which characterize native gingiva and PDL fibroblasts, to describe their function in tissue maintenance, and to discuss their possible modulation due to orthodontic tooth movement.Maintenance of the human periodontium requires the balance of proliferation and differentiation in the respective tissues' cells. Moreover, the cells must synthesize the extracellular matrix molecules and receptors that facilitate adhesion. To describe the molecules that contribute to periodontal tissue maintenance, we illustrate the localization of their expression and topography on frozen sections from native gingival tissue and primary cell cultures derived from periodontal ligament. In native gingival epithelium, proliferation is confined to basal and parabasal cells. Keratin K14, when used as structural marker, is visible in the entire epithelium, while K13, an indicator of early differentiation, is restricted to the suprabasal cell compartment. Vimentin indicates mesenchymal cells in the subgingival connective tissue. Concerning the matrix molecules, collagen type-IV is abundant at the epithelium-lamina propria interface, and fibronectin is apparent throughout the mesenchyme. The matrix receptor integrin beta1 reveals a pericellular localization in basal and parabasal cells, while focal adhesion kinase p125FAK is seen pericellularly in all epithelial layers. Cultures of primary periodontal ligament (PDL) fibroblasts (PDL-F) reveal expression of vimentin, strong proliferation, synthesis and extracellular deposition of collagen type-I and fibronectin. The integrin subunits beta1 and p125(FAK) are largely detectable at the cell periphery.     

复层牙周膜细胞膜片组织再生的实验性研究

目的:制备复层牙周膜干细胞膜片(MUCPs)与单层牙周膜干细胞膜片(MCPs),并评价其生物学活性和组织再生能力。方法:将构建的2种细胞膜片通过免疫组化和扫描电镜等方法观察其生物学活性。再将2种膜片分别与2种支架材料陶瓷牛骨(CBB)与预处理牙本质片(TDM)复合构建不同的生物性牙根,植入裸鼠皮下,观察组织再生情况。结果:体外结果显示MUCPs高表达Col-I、COL-III、Fibronectin、Laminin等细胞外基质的结构蛋白。体内结果显示在复层细胞膜片组可以观察到再生出更明显的类似于牙周复合组织的矿化沉积和牙周膜样纤维。结论:MUCPs具有更为良好的生物学活性和组织再生能力,将为临床牙周组织缺损的治疗提供一种新的治疗方法。     

Identification of a Fusobacterium nucleatum 65 kDa serine protease

A 65 kDa protease was partially purified from extracellular vesicles of Fusobacterium nucleatum cultures by preparative SDS-PAGE followed by electroelution. The pH optimum of the protease is 7.5-8.0 and its activity could be inhibited by serine protease inhibitors. The protease was found to degrade the extracellular matrix proteins fibrinogen and fibronectin as well as collagen I and collagen IV which were degraded at 37 degrees C but not at 28 degrees C, indicating the presence of a gelatinase activity in these bacteria. The 65 kDa protease was also able to digest the alpha-chains of immunoglobulin A but not immunoglobulin G. The 65 kDa F. nucleatum protease, capable of degrading native proteins, may play an important role in both the nutrition and pathogenicity of these periodontal microorganisms. The degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of periodontal tissues, and degradation of IgA may help the evasion of the immune system of the host by the bacteria.     

Emdogain regulation of cellular differentiation in wounded rat periodontium

Emdogain is an enamel matrix derivative that may promote periodontal regeneration by recapitulating critical events in tooth morphogenesis. We hypothesized that Emdogain enhances periodontal regeneration by promoting the differentiation of cells required for the synthesis of periodontal ligament, bone and cementum. Cell differentiation was examined in rat periodontal window wounds in which there is no microbial biofilm or epithelial downgrowth, thereby simplifying the model system. Defects were filled with vehicle control or Emdogain (3 mg/ml or 30 mg/ml). Rats were sacrificed at 7, 14 and 21 d after wounding. Specimens of periodontium were immunostained for osteopontin, bone sialoprotein, osteocalcin as markers of osteogenic differentiation and for alpha-smooth muscle actin, a myofibroblastic marker. Morphometry and 3H-proline radioautography were used for assessment of tissue homeostasis and matrix production. Rats treated with Emdogain (only at 30 mg/ml) showed widening of the periodontal ligament at 7 d; by 14 and 21 d, periodontal ligament width was restored to normal values for all groups. Emdogain exerted no effect on cementum thickness, bone volume, osteoid deposition rates, or extracellular staining for osteopontin, bone sialoprotein or osteocalcin. Further, the percentage of cells with intracellular staining for osteopontin, osteocalcin or bone sialoprotein was unaffected by Emdogain. Staining for alpha-smooth muscle actin was abundant in the repopulating wound but was also unaffected by Emdogain. In conclusion, Emdogain does not apparently affect the expression of differentiation markers or bone matrix protein synthesis in the repopulation response of wounded rat molar periodontium. Therefore the effect of Emdogain on wound healing in the periodontium may be independent of differentiation in the cell populations examined in this model.     

Promotion of functioning of human periodontal ligament cells and human endothelial cells by nerve growth factor

BACKGROUND: We have previously shown that cultured human periodontal ligament (HPL) cells produce nerve growth factor (NGF) and express mRNA of tyrosine kinase receptor (trkA), a high-affinity receptor of NGF. These findings suggest that NGF modulates the differentiation and proliferation of the periodontal ligament cells by paracrine and autocrine functions in vivo. Endothelial cells also express NGF and trkA. Therefore, NGF may regulate functions of periodontal ligament cells and endothelial cells during periodontal tissue regeneration. METHODS: Effects of NGF on expressions of bone/cementum-related proteins (osteocalcin [OC], bone sialoprotein [BSP], bone morphogenetic protein [BMP-7], core binding factor alpha [Cbfa-1], and type I collagen), calcification in HPL cells, and proliferation and mRNA expression of vascular endothelial growth factor (VEGF), an endothelial cell mitogen, in human microvascular endothelial cells (HMVECs) were examined. RESULTS: NGF elevated mRNA levels of OC, BSP, BMP-7, Cbfa-1, and type I collagen and enhanced mineral deposition in cultures of HPL cells. Furthermore, NGF stimulated mRNA expressions of VEGF-A and VEGF-B and cell proliferation in HMVEC. CONCLUSION: These findings suggest that the functional regulation of periodontal ligament cells and endothelial cells by NGF might result in the acceleration of periodontal tissue regeneration in vivo.     

Identification of a Fusobacterium nucleatum 65 kDa serine protease

A65 kDa protease was partially purified from extracellular vesicles of Fusobacterium nucleatum cultures by preparative SDS‐PAGE followed by electroelution. The pH optimum of the protease is 7.5–8.0 and its activity could be inhibited by serine protease inhibitors. The protease was found to degrade the extracellular matrix proteins fibrinogen and fibronectin as well as collagen I and collagen IV which were degraded at 37°C but not at 28°C, indicating the presence of a gelatinase activity in these bacteria. The 65 kDa protease was also able to digest the α‐chains of immunoglobulin A but not immunoglobulin G. The 65 kDa F. nucleatum protease, capable of degrading native proteins, may play an important role in both the nutrition and pathogenicity of these periodontal microorganisms. The degradation of extracellular matrix proteins by bacterial enzymes may contribute to the damage of periodontal tissues, and degradation of IgA may help the evasion of the immune system of the host by the bacteria.     

Flow cytometry analysis of guided tissue regeneration-associated human periodontal cells

BACKGROUND: Expanded polytetrafluoroethylene (ePTFE) barrier membranes have been widely used for guided tissue regeneration (GTR) of the human periodontal ligament (PL). However, the precise cellular and molecular events involved in the re-growth of the new tissue are still unclear. METHODS: Retrieved membranes and the newly-regenerated soft tissue (RT) underlying the membranes were used to examine the cells associated with GTR compared with normal human PL and gingival cells. Flow cytometry (FCM) was used, for the first time, to analyze the spindle-shaped fibroblast-like cells which were adherent to these membranes and the cells which grew out of the RT. RESULTS: The results showed that the membrane-associated (M) cells had the lowest rate of proliferation and appeared to be larger and more granular than the other types of cell. Moreover, both the M- and RT-derived cells were found to express higher levels of the extracellular matrix (ECM) proteins collagen type 1, fibronectin, tenascin, and decorin. In addition, evidence based on FCM profiles identified distinct sub-populations of GTR cells in which fibronectin expression was markedly up-regulated compared with normal PL cells and which also differed in size and granularity. CONCLUSIONS: The results of this study show that cells associated with GTR barrier membranes and with the underlying tissue appear to have distinct phenotypic and functional activities consistent with the production of new periodontal connective tissue and periodontal regeneration.