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The purpose of this study was to evaluate the effect of chlorhexidine and essential oils containing mouth rinses on oral wound healing after periodontal flap surgery. Eighty subjects participated in the study and were randomly assigned to use water, 0.12% chlorhexidine (CHX), essential oils (EO), 5% CHX, and 10% EO. Subjects were examined at 1, 2, and 3 weeks postoperatively. Plaque index (PI) and the modified gingival index (GI) were recorded, while wound epithelialization was measured to evaluate the healing process. Numerical data were analyzed with parametric test for multiple comparisons (ANOVA) with Bonferroni correction. Categorical data were analyzed using Chi-square test/fisher exact test. All groups demonstrated a gradual GI reduction from first to third visit. Patients in the CHX group presented statistically significant lower PI scores than patients in the water group at the all-time points of the study. Wound epithelialization analysis demonstrated that 100% of the sites in the CHX group were healing by secondary intention at visit 1. This finding was statistically significant. Full strength concentrations of CHX and EO did not show any detrimental effects on healing after traditional periodontal surgery at the end of the observation period. The use of chlorhexidine and EO containing mouthwashes does not appear to delay wound healing. Diluting these commercial mouthwashes may present an approach that could possibly reduce the adverse effects (such as tooth staining) associated with their use, while maintaining their antibacterial properties.

Introduction Endocyn, a pH-neutral solution of hypochlorous acid and hypochlorite has been developed for use as an endodontic irrigant. The purpose of this study was to evaluate the effect of Endocyn on human periodontal ligament (PDL) fibroblasts, rat osteosarcoma cells (UMR-106), and stem cells of the apical papilla (SCAP) compared with other commonly used endodontic irrigants. Methods To determine cytotoxicity, cells were exposed to various concentrations of Endocyn, 6% sodium hypochlorite (NaOCl), 17% EDTA, and 2% chlorhexidine for 10 minutes, 1 hour, or 24 hours. Cell survival was measured fluorescently using calcein AM. Endocyn also was tested for its ability to inhibit SCAP proliferation and alkaline phosphatase activity. Finally, SCAP transcript expression was examined via reverse-transcriptase polymerase chain reaction. Results Endocyn was no more toxic to PDL and UMR cells than water for up to 24 hours. Endocyn concentrations of 50% were toxic to SCAP after 1 hour of exposure. Endocyn concentrations of >20% inhibited SCAP proliferation, whereas concentrations of ≥10% inhibited alkaline phosphatase activity. Exposure of SCAP to 10% Endocyn for 3 days did not alter most transcript expression, but did significantly reduce the expression of alkaline phosphatase, fibromodulin, and osteomodulin. Conclusion Endocyn was significantly less cytotoxic to PDL, UMR-106, and SCAP cells compared with other commonly used endodontic irrigants. High concentrations of Endocyn did inhibit some transcript expression and alkaline phosphatase activity, indicating a potential reduction in the osteogenic potential of stems cells exposed to Endocyn.

Smokers have an increased incidence and severity of periodontal disease. Although cigarette smoke contains4,000 chemical components that could affect periodontal tissues, less is understood about the effect of smokeless tobacco. Therefore, this study compares the effects of cigarette smoke extract (CSE) and smokeless tobacco extract (STE) on cell survival and motility of periodontal ligament (PDL) and gingival fibroblasts in vitro.PDL and gingival fibroblasts were exposed to various concentrations of CSE, STE, or nicotine alone. Viable cells were labeled with calcein acetoxymethyl, visualized using fluorescent microscopy, and quantified using a fluorescence multi-well plate reader. In vitro wounding and collagen gel contraction assays were used to assess cell motility.Both gingival and PDL fibroblasts displayed reduced cell viability with increasing concentrations of CSE and STE. Based on relative nicotine content, CSE was significantly more cytotoxic than STE. PDL fibroblasts were also more sensitive to both CSE and STE compared with gingival fibroblasts. Finally, sublethal doses of CSE reduced cell motility and gel contraction, whereas STE had less effect. Nicotine alone ≤0.5 mM had little to no effect in any of these assays.Many of the underlying effects of tobacco products on periodontal tissues may be due to direct inhibition of normal fibroblast function. CSE is found to be more deleterious to the function of both PDL and gingival fibroblasts than STE. PDL fibroblasts appear to be more sensitive to CSE and STE than gingival fibroblasts. Therefore, cigarette smoke may have more profound effects than smokeless tobacco.

To investigate the effect of silver diamine fluoride (SDF) and fluoride varnish (FV) on human gingival fibroblasts (HGF) and bacteria.HGF cell viability was assessed after exposure to various dilutions of SDF or FV. Hydroxyapatite (HA) discs treated with SDF, FV, or saline were rinsed in artificial saliva for 84 days. HGF were exposed to treated discs and viability assessed fluorescently. Oral bacteria were exposed to treated discs and survival quantified.At 0.01%, SDF was almost 100% cytotoxic to HGF. SDF and FV treated HA discs, induced near-complete cell death after 24 hours of contact. After rinsing FV discs for 21 days, cell survival exceeded 95%. SDF treated discs were toxic to HGF and bacteria after 9 weeks of rinsing.SDF and FV can induce cell death. FV lost its cytotoxicity within 3 weeks, while SDF remained cytotoxic even after 9 weeks of rinsing. This research confirms that SDF has long lasting antimicrobial effects at very low concentrations although it does raise concerns regarding cytotoxicity. However, HGF cells are exposed to other cytotoxic substances in dentistry with little, if any, long-term effects.

The toxic effects of cigarette smoke often presents in smokers as increased incidence and severity of periodontal disease. These patients demonstrate symptomatic inflammation, increased probing depth, and tooth loss likely attributable to the direct effects of cigarette smoke on periodontal ligament (PDL) fibroblasts. The goal of this in vitro study is to investigate the direct effects of smoking on PDL fibroblasts, focusing on cell-extracellular matrix (ECM) interactions and cell survival.PDL cells were plated for various times on tissue culture plastic, PDL-derived ECMs, collagen Type I, or fibronectin. Cells were exposed to various concentrations of cigarette smoke extract (CSE) at different times during the cell attachment process. Subsequently, cell survival was quantified using calcein-acetoxymethyl ester compound and a fluorescent plate reader.After exposure to CSE, PDL cell survival increased with increased cell attachment time to plastic. These observations were independent of soluble factors present in PDL cell-conditioned media. PDL-derived ECMs and collagen Type I-pretreated plates promoted increased cell survival after 1 day of cell attachment. Fibronectin-pretreated plates demonstrated increased cell survival after 3 days of cell attachment.Cell-ECM interactions increase survival of PDL cells exposed to CSE. It is suggested that the increased survival is attributable to PDL cells altering their ECM, potentially by depositing collagen and fibronectin. This may imply that cells embedded in an ECM would be more resistant to the toxic effects of cigarette smoke, leading to increased cell death near the exposed edges of a wound.

Native zein electrospun nanofibers have shown poor solvent resistance and low mechanical strength. Compared to other toxic cross-linkers, a safer method of stabilizing zein based fibers while retaining or with improved mechanical strength is needed to convert these materials for biomedical applications where culture media or body fluids may be present. We report here a method of fabricating non-toxic zein nanofibers using reactive electrospinning coupled with in situ photo-cross-linking. The cross-linked zein nanofibers exhibited significantly improved mechanical strength and sustained morphology against water and aqueous ethanol solution. This process doesn't require additional conventional cross-linking agents to form cross-linking network, which is advantageous for biomedical applications. Antimicrobial monomer with photo-reactive moiety was coupled with methacrylate zein nanofibers and showed strong inhibitory activity against cariogenic Streptococcus mutans. Cytotoxicity test with human gingival fibroblasts revealed high biocompatibility.

The fungal pathogen Candida albicans causes a variety of oral infections, including denture stomatitis, which is characterized by inflammation of the oral mucosa in direct contact with dentures and affects a significant number of otherwise healthy denture wearers. While antifungal treatment reduces symptoms, infections are often recurrent. One strategy to address this problem is to incorporate compounds with fungicidal activities into denture materials to prevent colonization. Our laboratory synthesized novel derivatives of 1,4-diazabicyclo[2.2.2]octane (DABCO), which is an organic compound typically used as a catalyst in polymerization reactions. DABCO derivatives with different aliphatic chain lengths (DC16, DC16F, DC18, and C6DC16), as well as methacrylate monomers conjugated to DABCO compounds (DC11MAF and C2DC11MAF), were synthesized and tested for antimicrobial activity. All the compounds exhibited fungicidal activity against several Candida species at concentrations ranging between 2 and 4 μg/ml. Moreover, acrylic denture base resins fabricated to contain 1, 2, or 4 wt% DABCO compounds inhibited surface C. albicans biofilm formation, as well as fungal growth, in disc diffusion assays. Remarkably, discs (4 wt%) aged for 2 months also exhibited approximately 100% growth-inhibitory activity. While some DABCO compounds exerted intermediate to high cytotoxicity against mammalian oral cell types, DC11MAF and denture base resin discs containing 2 or 4 wt% C2DC11MAF exhibited relatively low cytotoxicity against periodontal ligament (PDL) cell and gingival fibroblast (GF) lines, as well as primary oral epithelial cells. These studies demonstrate that DABCO derivatives can be incorporated into denture materials and exert fungicidal activity with minimal cytotoxicity to mammalian cells. DC11MAF and C2DC11MAF are considered strong candidates as therapeutic or preventive alternatives against Candida -associated denture stomatitis.

The expansion of evidence-based dentistry (EBD) is essential to the continued growth and development of the dental profession. Expanding EBD requires increased emphasis on critical thinking skills during dental education, as noted in the American Dental Education Association's Competencies for the New General Dentist. In order to achieve this goal, educational exercises must be introduced to increase the use of critical thinking skills early in the dental curriculum, with continued reinforcement as students progress through subsequent years. Described in this article is one approach to increasing student exposure to critical thinking during the early basic science curriculum-specifically, within the confines of a traditional histology course. A method of utilizing the medical and dental research literature to reinforce and enliven the concepts taught in histology is described, along with an approach for using peer-to-peer presentations to demonstrate the tools needed to critically evaluate research studies and their presentation in published articles. This approach, which could be applied to any basic science course, will result in a stronger foundation on which students can build their EBD and critical thinking skills.

Introduction The purpose of this in vitro study was to compare the biocompatibility of a novel formulation of a silicone-based endodontic sealer GuttaFlow 2 (GF2; Coltene/Whaledent, Langenau, Germany) with the original (GFO) and fast-set (GFF) formulations of GuttaFlow and with an epoxy resin sealer, AHPlus Jet (AH+J; Dentsply, York, PA). Methods Sealers were set into 3 × 5.5 mm discs. Cell culture media was used to extract leachable products at 24 hours and 1, 2, and 4 weeks. Primary human periodontal ligament fibroblasts were incubated with sealer elutes for 24 hours and evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay and the calcein AM assay. Cell attachment was evaluated on set sealer that was either rinsed or unrinsed with cell media for 1 week. Statistical analysis was performed using the Student t test. Results Both calcein and 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays revealed that periodontal ligament cell viability was reduced on AH+J at 1, 2, and 4 weeks compared with all GuttaFlow sealers. There were no differences in cell viability between the GuttaFlow samples, and all displayed high rates of cell survival at all time periods. After 2 hours, cell attachment to the rinsed GFO and GFF samples exceeded the control, and at 24 hours cell attachment on all GuttaFlow samples exceeded the control. AH+J sealers supported significantly less cell attachment when compared with all GuttaFlow sealers. Cell attachment to set sealers showed better cell attachment when rinsed compared with unrinsed. Conclusions GuttaFlow sealers were more biocompatible than AHJ in vitro. The novel GF2 displayed comparable biocompatibility with GFF and GFO.

Chemical plaque control is the most commonly recommended means of oral hygiene after periodontal surgery. Commercially available mouthwashes contain a variety of active ingredients that have bactericidal properties but may potentially be toxic to the host cells. The goal of this in vitro study is to investigate the effect of commercially available mouthwashes on the survival and migratory capacity of human fibroblasts.Human gingival and periodontal ligament (PDL) fibroblasts were treated with commercially available mouthwashes that contained either chlorhexidine (CHX) or essential oils (EO) as the active ingredient. Each mouthwash was tested over a range of concentrations for its ability to affect fibroblast survival and migration, as well as long-term effects on cell viability.Undiluted mouthwashes induced near-complete cell death 24 hours after only a 60-second treatment. Dilutions of 15% to 20% for both CHX and EO mouthwashes resulted in 50% cell death. When diluted to 10% to 15%, EO did not reduce cell migration, whereas similar dilutions of CHX resulted in reduced cell migration. Concentrations of 10% of both EO and CHX mouthwashes retained most of their antibacterial capacity. Treatment with EO did not result in gingival fibroblast death, whereas 5% CHX resulted in near-complete gingival fibroblast death 7 days after exposure.The results of this in vitro study indicate that diluted EO displayed no detectable detrimental effects on human gingival and PDL fibroblasts, whereas diluted CHX reduced both cell migration and long-term survival. Both solutions retained their antimicrobial activity in lower concentrations.

Introduction Fractured endodontic files present a major problem. A novel method has been proposed to retrieve fractured nickel-titanium (NiTi) endodontic files by using electrochemical dissolution. However, the effect of file dissolution on adjacent soft tissues such as the periodontal ligament (PDL) has not been investigated. The aim of this study was to determine the effects of the dissolution products on PDL fibroblasts. Methods Endodontic files were dissolved in sodium fluoride (NaF) by passing a 50-mA current through the NiTi files while immersed in the NaF solution. NaF/NiTi solutions were diluted with minimal essential medium-α media containing 10% serum. PDL cells were treated for up to 24 hours, and cell viability was quantified by using calcein AM to label live cells and ethidium homodimer to label dead cells. This was repeated by using artificial saliva (AS) as an alternative to NaF. Results NaF solution reduced PDL cell survival, and the NaF/NiTi solution further reduced PDL cell survival. AS alone did not reduce cell survival, whereas AS/NiTi solution reduced PDL cell survival. Particles that resulted from the electrochemical dissolution of NiTi files were highly cytotoxic. Conclusions Electrochemically dissolving NiTi files in NaF results in solutions that are cytotoxic to PDL fibroblasts. AS may be a less toxic alternative for dissolving NiTi files.

The search still continues to find the best storage media for avulsed teeth. Unfortunately, some of the recommended storage solutions are not commonly found in households or do not preserve the periodontal ligament (PDL) cells long-term. The purpose of the present study was to determine whether Pedialyte is a viable alternative storage solution for avulsed teeth by assessing its ability to preserve human PDL cell viability.Human PDL cells were exposed to 6 different storage solutions (minimal essential medium [MEMα], Hank's balanced salt solution [HBSS], non-fat milk, coconut water, Pedialyte, or tap water) for 2, 6, 24, or 48 hours at 4°C or 25°C. Cell viability was quantified immediately or 1 week after exposure. The effects of these storage solutions on PDL cell motility and bacterial proliferation were also examined. The results were statistically analyzed by analysis of variance.Pedialyte at 4°C and 25°C showed significantly (P.001) higher cell survival compared with water after all time intervals. No significant difference was noted between control (MEMα), HBSS, coconut water, and Pedialyte at 4°C after 2 hours. Cells stored in Pedialyte for 24 hours at 25°C and assayed 1 week later showed significantly higher cell survivability compared with milk. Pedialyte supported significantly less bacterial growth compared with non-fat milk and coconut water. No difference in cell motility was observed for cells stored for 24 hours in Pedialyte, MEMα, HBSS, milk, or coconut water.Pedialyte is a viable alternative as a storage solution for avulsed teeth.

Background: The periodontal ligament (PDL) is the connective tissue that anchors the cementum of the teeth to the alveolar bone. PDL fibroblasts are responsible for the production of collagen and remodeling of the PDL. Periodontal disease is increased among smokers in both incidence and severity. This study examines the direct effect of smoking on PDL fibroblasts and their production of various matrix components and remodeling enzymes. Methods: PDL cells were plated for 1 day and then treated with various concentrations of cigarette smoke extract (CSE). Survival of PDL cells was quantified after exposure to CSE, and their ability to contract three-dimensional collagen gels was examined. Changes in transcript expression after CSE treatment was compared using reverse transcription-polymerase chain reaction analysis for matrix metalloproteinases (MMPs), collagens, and integrins. Results: Treatment with CSE-induced cell death at concentrations of ‡5%. PDL-cell-induced collagen gel contraction was reduced at concentrations of 1.5% CSE. Treatment with CSE selectively increased the expression of collagen Va3 and decreased collagen XIa1. CSE increased the expression of MMP1 and MMP3 and, to a lesser extent, MMP2 and MMP8. CSE also increased the expression of integrins a1, a2, and a10 (collagen receptors) and a9 (a tenascin receptor). Conclusions: This study shows that cigarette smoking has local effects on the cells of the PDL. CSE reduced survival of PDL cells and their ability to contract collagen matrices. CSE also altered the expression of molecules known to provide the structural integrity of the ligament by altering collagen synthesis and remodeling as well as cell adhesion. J Periodontol 2012;83:787-796.

Periodontal remodeling requires coordinated cell movement. Semaphorins are cell-surface signals that regulate cell migration and may be differentially regulated by periodontal cells. Mechanical tension can regulate periodontal ligament (PDL) remodeling. We predicted that mechanical tension alters the expression of the subset of semaphorins in the periodontium likely to be most involved with regulating the remodeling of this tissue.PDL and gingival cells were exposed to mechanical tension, and their attachment and movement on collagen matrices were evaluated. Alterations in extracellular matrix and semaphorin transcript expression were monitored by semiquantitative reverse transcription-polymerase chain reaction.Mechanical tension induced osteoclast regulatory transcripts in the PDL cells to a greater extent than gingival fibroblasts, increasing the expression of osteoprotegerin and decreasing receptor activator of nuclear factor-kappa B ligand. These mechanical forces reduced PDL cell mingling, without altering cell attachment or motility. Concurrently, these forces induced dynamic changes in several semaphorin molecules in PDL cells, increasing semaphorin 3D and 5B and decreasing semaphorin 7A. In addition, plexin transcript expression was altered, decreasing plexin A1 and increasing plexin C1. These changes were different than those observed in gingival fibroblasts.These data suggest that a subset of semaphorins and plexins are dynamically regulated in the PDL. Because these molecules may be involved in cell guidance, changes in semaphorins may play a pivotal role in periodontal remodeling, affecting angiogenesis or PDL cell invasion into sites of injury.

During periodontal regeneration, multiple cell types can invade the wound site, thereby leading to repair. Cell motility requires interactions mediated by integrin receptors for the extracellular matrix (ECM), which might be useful in guiding specific cell populations into the periodontal defect. Our data demonstrate that fibroblasts exhibit differential motility when grown on ECM proteins. Specifically, gingival fibroblasts are twice as motile as periodontal ligament fibroblasts, whereas osteoblasts are essentially non-motile. Collagens promote the greatest motility of gingival fibroblasts in the following order: collagen III>collagen V>collagen I. Differences in motility do not correlate with cell proliferation or integrin expression. Osteoblasts display greater attachment to collagens than does either fibroblast population, but lower motility. Gingival fibroblast motility on collagen I is generally mediated by alpha2 integrins, whereas motility on collagen III involves alpha1 integrins. Other integrins (alpha10 or alpha11) may also contribute to gingival fibroblast motility. Thus, ECM proteins do indeed differentially promote the cell motility of periodontal cells. Because of their greater motility, gingival fibroblasts have more of a potential to invade periodontal wound sites and to contribute to regeneration. This finding may explain the formation of disorganized connective tissue masses rather than the occurrence of the true regeneration of the periodontium.

Periodontal regeneration requires the coordinated movement and differentiation of several cell types in order to re-establish the cementum, periodontal ligament (PDL), and alveolar bone. Cells in culture are often used as model systems for mature tissues, although they may represent expanded progenitor cell populations. Comparison of transcript expression between fresh PDL tissue and PDL cell isolates by MicroArray analysis has revealed numerous molecular differences. Several transcripts (including alkaline phosphatase, bone sialoprotein, periostin, and fibromodulin) are expressed at higher levels in fresh PDL than in cultured PDL cells. In contrast, PDL cells in culture selectively express a variety of growth factors. Several of these growth factors alter PDL fibroblast behavior. Two members of the transforming growth factor beta family of growth factors, namely, bone morphogenic protein-7 (BMP7) and growth differentiation factor-5 (GDF5), reduce cell proliferation and Stro-1 expression (a bone marrow stromal stem cell marker), whereas only BMP7 induces alkaline phosphatase activity. In contrast, fibroblast growth factor-5 induces enhanced cell proliferation and Stro-1 expression, while repressing alkaline phosphatase activity. The stimulation of PDL cells to differentiate (either by BMP7 or GDF5) inhibits cell motility. Thus, PDL cells in culture are regulated by several factors that differentially stimulate a mineralized (cementoblast-like) fate, a non-mineralized fate (mature fibroblasts), or the propagation of a more naive phenotype (potential progenitors).

Fibroblasts are critical to the establishment and maintenance of the periodontal attachment apparatus (cementum, periodontal ligament [PDL], and bone). In order to characterize the cellular changes that accompany periodontal regeneration, better tools are necessary to distinguish periodontal ligament fibroblasts (PDLF), gingival fibroblasts, and osteoblasts. Our goal is to identify gene markers to better characterize and identify these cell types.We chose to examine and compare the expression of numerous gene transcripts by semiquantitative reverse transcriptase-polymerase chain reaction using primers specific for 44 different gene transcripts in order to better characterize the identity of these cells.Several transcripts were cell-type specific. Specifically, fibromodulin was expressed only in PDL fibroblasts, while osteopontin was expressed only in dermal fibroblasts. In addition, lumican was expressed by all three types of fibroblasts (PDL, gingival, and dermal), while alkaline phosphatase was expressed by osteoblasts as well as PDL and gingival fibroblasts.Our results indicate that PDL fibroblasts are distinct from either gingival or dermal fibroblasts or osteoblasts. In general, PDL and gingival fibroblasts displayed greater similarity to each other than either displayed toward dermal fibroblasts. Furthermore, both gingival and PDL fibroblasts displayed greater similarity to osteoblasts than to dermal fibroblasts, possibly reflecting their common origin (the neural crest).

Clinical studies using locally applied doxycycline hyclate (DHV) have demonstrated significant probing depth reduction and gain in clinical attachment as a monotherapy without scaling and root planing. The mechanism for this attachment level gain to the non-root planed tooth is not understood. The purpose of this study was to investigate the effect of locally applied doxycycline hyclate on human gingival fibroblast attachment to subgingival calculus on contaminated root surfaces.Two separate experiments were performed, both on subgingival calculus. In experiment 1, teeth with subgingival calculus were treated with either doxcycycline hyclate in bioabsorbable vehicle (DHV) or with vehicle control (VC) in vivo. In experiment 2, teeth with subgingival calculus were treated with DHV, VC, scaling and root planing (SRP), or no treatment in vitro. The amount of cell attachment to calculus-covered root surfaces was quantitatively compared using a fluorescent dye assay and epifluorescence microscope. Values for cell attachment are presented as the mean standard deviation of the mean. The data were evaluated using Student t test.In both experiments, there was no statistically significant difference in fibroblast attachment in the DHV, VC, or no treatment groups (P0.05). The SRP group showed significantly more cellular attachment to tooth surfaces formerly covered by subgingival calculus than all other groups (P0.001). In general, more cells attached to cementum than to calculus. Root chips that showed no attachment to the subgingival calculus also had no cells attached to the adjacent cemental root surface.The addition of doxycycline hyclate in a bioabsorbable vehicle used as a locally delivered drug did not enhance the initial cellular attachment of human gingival fibroblasts to subgingival calculus or contaminated root surfaces.

The expansion of evidence-based dentistry (EBD) is essential to the continued growth and development of the dental profession. Expanding EBD requires increased emphasis on critical thinking skills during dental education, as noted in the American Dental Education Association's Competencies for the New General Dentist. In order to achieve this goal, educational exercises must be introduced to increase the use of critical thinking skills early in the dental curriculum, with continued reinforcement as students progress through subsequent years. Described in this article is one approach to increasing student exposure to critical thinking during the early basic science curriculum-specifically, within the confines of a traditional histology course. A method of utilizing the medical and dental research literature to reinforce and enliven the concepts taught in histology is described, along with an approach for using peer-to-peer presentations to demonstrate the tools needed to critically evaluate research studies and their presentation in published articles. This approach, which could be applied to any basic science course, will result in a stronger foundation on which students can build their EBD and critical thinking skills.

Bone replacement graft (BRG) materials are used in periodontal therapy to encourage new bone formation. Extracellular matrix proteins may improve periodontal ligament fibroblast (PDLF) attachment to these materials. We demonstrate that PDLFs adhere well to the extracellular matrix (ECM) proteins fibronectin, vitronectin, laminin, and collagen types I and IV. PDLFs express numerous ECM-receptor integrin subunit transcripts (α1, a2, a3, a4, a5, α11, (31, β5, and (38) at high levels, while others (a6, a9, aV, β3, (36, and β7) are expressed at reduced levels. Despite the fact that PDLFs adhere well to fibronectin and collagen type IV bound to plastic, and express integrins that recognize these ECM proteins, they do not attach well to anorganic bovine bone matrix (ABM) coated with these same proteins. However, the addition of vitronectin, laminin, or collagen type I to these same ABMs substantially increased PDL cell attachment. Thus, selective use of ECM proteins may be clinically useful in promoting cell attachment to ABM and bone regrowth. Abbreviations: extracellular matrix (ECM), periodontal ligament (PDL), bone replacement graft (BRG), anorganic bovine bone matrix (ABM), reverse transcriptase polymerase chain-reaction (RT-PCR).

Bone replacement graft (BRG) materials are often used to treat periodontal defects, to promote cellular invasion, and to encourage bone regrowth. Periodontal ligament fibroblasts (PDLF) incorporate these materials and form the basis of the renewed connection between the existing and newly formed alveolar bone and the tooth surface. A peptide (P-15) that mimics the putative cell-binding domain of collagen has been reported to promote dermal fibroblast attachment and proliferation.PDLF were quantitatively examined for their ability to adhere to a variety of BRG materials fluorometrically. In addition, scanning electron microscopy was used to examine the changes in morphology exhibited by these cells as they attached and spread on several BRG materials. Finally, BRG materials containing the P-15 peptide were quantitatively examined for their ability to promote PDLF attachment and proliferation.Freeze-dried allograft bone supports greater PDLF attachment than does several xenograft and alloplastic anorganic bone replacement materials. An anorganic BRG material containing the P-15 peptide promoted more rapid cell attachment and spreading than a similar anorganic BRG material lacking this peptide. Finally, none of the BRG materials examined promoted PDLF proliferation.Our data indicate that the addition of the P-15 peptide increases the rapidity of PDLF attachment to xenogeneic bone replacement materials. This increase in the rate of attachment may have clinical significance in the context of the dynamic regulation of cell attachment during periodontal regeneration. However, this peptide does not promote an increase in stable cell attachment or proliferation in vitro.

Fibroblasts are the predominant cells of the periodontal ligament and the gingiva and have important roles in the function and regeneration of the tooth support apparatus. The goal of this study was to investigate the possible differences in the adhesion properties and expression of extracellular matrix (ECM) receptors among different fibroblast populations.The adhesion of gingival (GF), dermal (DF), and periodontal ligament fibroblast (PDLF) cultures to ECM proteins (fibronectin, laminin, vitronectin, RGD peptide, collagen type I, and collagen type IV) adsorbed to tissue culture plastic was evaluated fluorometrically. Quantitative reverse transcription-polymerase chain reactions (RT-PCR) were performed using primers specific for 19 integrin subunits to quantify ECM receptor transcript expression.Our data demonstrated that GF and PDLF adhere to vitronectin and collagen types I and IV more avidly than do DF. PDLF adhered well to laminin, whereas GF and DF did not. Quantitation of integrin expression demonstrated that the different fibroblast types expressed different integrin transcripts, further demonstrating their innate differences.The 3 fibroblast types studied behave differently and expressed different ECM receptors. However, gingival fibroblasts and periodontal ligament fibroblasts are more similar in their attachment and integrin expression than either is to dermal fibroblasts. Therefore, experiments using DF will not necessarily be valid for oral tissues.

Cellular interactions with laminin are important for numerous morphogenetic events. In Xenopus, the first of these is neurulation. The integrin alpha6 subunit mediates an attachment of the cells of the neural plate to the underlying basal lamina. A disruption of this interaction results in embryos that fail to neurulate (T. E. Lallier et al., 1996, Development 122, 2539-2554). Here we provide evidence supporting the specificity of this phenomenon and characterize developmental events as either disrupted or unaffected by a perturbation of alpha6 integrin expression. First, reduction of alpha6 integrin expression does not halt mitotic division throughout the embryo, indicating that the neural defects observed are not simply a global perturbation of all developmental processes. Second, a gene associated with dorsal mesoderm formation, brachyury, is expressed normally in alpha6 integrin-perturbed embryos. Third, the expression of BMP4, noggin, chordin, and follistatin, all of which are critical for neural induction, are at near normal levels. In addition, several genes expressed shortly after neural induction (N-CAM, nrp1, and Xanf1) are not perturbed in nonneurulating embryos. Interestingly, expression of one neural-specific gene (synaptobrevin), which is normally detectable late in neurulation, is abolished in these alpha6 integrin-perturbed embryos. Furthermore, the spatial expression of several transcripts is expanded in alpha6 integrin-perturbed embryos (orthodenticle and engrailed). Taken together, these data indicate that while alpha6 integrin-mediated interactions with laminin are required for neurulation, they are not required for the initial processes of neural induction. However, these cell-extracellular matrix interactions appear to be important in later inductive events and rostrocaudal patterning of the neural tube.

Recently, the use of recombinant full-length amelogenin protein in combination with fluoride has shown promising results in the formation of densely packed enamel-like structures. In this study, amelogenin (rP172)-releasing hydrogels containing calcium, phosphate, and fluoride were investigated for remineralization efficacy using in vitro early enamel caries models. The hydrogels were applied to artificial caries lesions on extracted human third molars, and the remineralization efficacy was tested in different models: static gel remineralization in the presence of artificial saliva, pH cyclic treatment at pH 5.4 acetic buffer and pH 7.3 gel remineralization, and treatment with multispecies oral biofilms grown in a continuous flowing constant-depth film fermenter. The surface microhardness of remineralized enamel increased significantly when amelogenin was released from hydrogel. No cytotoxicity was observed when periodontal ligament cells were cultured with the mineralized hydrogels.

A new dimethacrylate chelating monomer containing a BisGMA-like backbone structure and a bis(carboxymethyl)-L-lysine chelating group and its ternary zirconium-fluoride complex (antibacterial fluoride-releasing monomer) have been synthesized. The monomer structures were confirmed by (1)H-NMR, (13)C-NMR, and ES-MS analysis. Several experimental fluoride-releasing dental composites containing different quantities of the new antibacterial fluoride-releasing monomer were formulated and tested for fluoride release, fluoride recharge, compressive and flexural strengths, water sorption and solubility. These composites displayed high fluoride release and recharge capabilities, as well as good physical and mechanical properties.

Titanium implants are widely used in dentistry to replace lost teeth. Various surface modifications have been used to improve implant retention and osseointegration. This study is designed to compare the ability of three titanium surfaces to promote cell attachment and cell motility of cells relevant to periodontal tissues.Three clinically relevant surfaces were tested: 1) machined titanium; 2) a titanium surface roughened through acid etching (dual thermal-etched titanium [DTET]); and 3) a titanium surface roughened with nanometer-scale calcium phosphate deposition (nanoscale calcium phosphate-impregnated titanium [NCPIT]). Cell attachment and migration were examined for four cell types: rat osteosarcoma cells, human osteoblasts, and gingival and periodontal ligament (PDL) fibroblasts.All four cell types attached to each of the three titanium surfaces equally by 2 hours, and the PDL and gingival fibroblasts generally displayed less attachment than the osteosarcoma cells and osteoblasts. The cells displayed differential motility and long-term attachment to each of the titanium surfaces. Osteosarcoma cells displayed preferential motility on NCPIT, whereas PDL fibroblasts were more motile on machined titanium, and gingival fibroblasts moved more rapidly on both DTET and NCPIT. Osteoblasts displayed little motility on any of the titanium surfaces and lost viability on NCPIT after 24 hours. Gingival fibroblasts lost attachment to machined titanium.Periodontal cells displayed differential motility and long-term attachment to titanium surfaces. Selective modification of titanium surface properties in various regions of an implant may be useful in guiding specific cell populations to specific locations where they might best aid in osseointegration and soft tissue remodeling.

Cells of the periodontal attachment (cementoblasts, osteoblasts, and periodontal ligament fibroblasts) are descended from a common progenitor (the cranial neural crest). During their differentiation into different cell types, these cells separate from one another to form a laminated structure. Semaphorins (and their neuropilins and plexin receptors) act as cell guidance molecules for other neural crest derivatives. It is predicted that the differential expression of these molecules will correlate with the ability of these cells to segregate. It is demonstrated that human pre-osteoblasts segregate from PDL and gingival fibroblasts in culture. In addition, these cells express different semaphorins and plexins. Semaphorins 3D and 7A were expressed preferentially in dermal fibroblasts, while semaphorin 6B was selectively expressed by pre-osteoblasts. Semaphorins 3B, 4C, 5B, and 6C and plexins B1 and C1 were expressed in reduced levels in pre-osteoblasts. Analysis of the data suggests that differential expression of semaphorins and plexins may be involved in regulating cell-sorting in the formation and regeneration of the periodontal attachment structure. Abbreviations: Periodontal Ligament (PDL), Reverse Transcriptase Polymerase Chain-reaction (RT-PCR).

This study evaluated the attachment of cultured explants of human periodontal ligament fibroblasts and gingival fibroblasts to different root-end-filling materials. Although periodontal ligament and gingival fibroblasts initially attached avidly to Geristore, these same cells displayed no significant attachment to ProRoot, Tytin amalgam, or SuperEBA. With further incubation on Geristore, the attachment of both periodontal ligament and gingival fibroblasts improved and these cells proliferated. In contrast, no improvement in attachment or proliferation was observed for cells incubated for greater times with ProRoot, Tytin amalgam, or SuperEBA. Because the attachment characteristics of these two groups of fibroblasts were identical, we examined the potential role of the extracellular matrix family of receptors (integrins) on the attachment of gingival fibroblasts. Gingival fibroblast attachment to collagen type I was determined to be dependent on alpha1beta1 and alpha2beta1 integrins, whereas their attachment to the RGD-binding sequence of fibronectin and vitronectin was partially inhibited by antibodies to the beta1 and alphaV integrin subunits. However, attachment of gingival fibroblasts to Geristore was not reduced by the addition of any of the attachment-perturbing anti-integrin antibodies examined. Thus, gingival fibroblasts attach to Geristore, but this attachment was mediated by mechanisms other than integrins.

Background: Regenerative periodontal treatment aims to restore the attachment of the periodontal ligament and gingival collagen fibers to both the cementum of the root surface and alveolar bone. Fibroblasts are the predominant cells of the periodontal ligament and gingiva and have important roles in the function and regeneration of the tooth-supporting apparatus. This study investigated whether a putative collagen-based cellbinding peptide (P-15) increases gingival fibroblast attachment to root shavings and bone replacement graft (BRG) materials. Methods: Gingival and dermal fibroblast attachment to root shavings and BRG materials, and cell proliferation on root shavings and sections were measured fluorometrically. Root shavings and root sections obtained from periodontally healthy teeth were treated with P-15 at 2 concentrations (200 ng/g or 400 ng/g). Citric acid (CA)-treated root materials were also compared to untreated root shavings and root sections that served as negative control groups. Results: Attachment of all cells to bone fragments (whether freeze-dried or demineralized) was significantly greater than to hydroxyapatite (HA)-based BRG materials. The addition of P-15 to HA did not significantly increase gingival or dermal fibroblast attachment. At a concentration of 400 ng/g, P-15 significantly increased gingival and dermal fibroblast attachment to root shavings as compared to untreated shavings. Bone fragments, HA-based BRG materials, and untreated root shavings inhibited gingival fibroblast proliferation. Treatment of root sections with P-15 did not have any effect on gingival fibroblast proliferation. Conclusions: P-15 is a potential alternative to CA for promoting fibroblast attachment to root surfaces. However, P-15 did not enhance fibroblast proliferation on root sections. J Periodontol 2003;74:458-467.

To examine the role of the lipid mediator platelet-activating factor (PAF) in epithelial wound healing.A 7-mm central de-epithelializing wound was produced in rabbit corneas, and the tissue was incubated with 125 nM carbamyl PAF (cPAF), an analogue of PAF. Rabbit corneal epithelial and stromal cells were also cultured in the presence of cPAF. Cell adhesion, proliferation, and migration assays were conducted. Apoptosis was assayed by TUNEL staining on preparations of corneal tissue sections and in cells in culture.Twenty-four hours after injury, 50% of the wounded area was covered by new epithelium, whereas only 30% was covered in the presence of cPAF. At 48 hours, the epithelium completely closed the wound, but only 45% of the original wound was covered in corneas treated with cPAF. Similar inhibition of epithelial wound closure was found with human corneas incubated with PAF in organ culture. Moreover, addition of several growth factors involved in corneal wound healing, such as epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, could not overcome the inhibitory action of PAF in wound closure. Three PAF antagonists, BN50727, BN50730, and BN50739, abolished the effect of PAF. A significant increase in TUNEL-positive staining occurred in corneal stromal cells (keratocytes), which was inhibited by preincubating the corneas with PAF antagonists. However, no TUNEL-positive staining was found in epithelial cells. TUNEL-staining results in cultured stromal cells (keratocytes) and epithelial cells in first-passage cell culture were similar to those in organ-cultured corneas. In addition, PAF caused 35% to 56% inhibition of adhesion of epithelial cells to proteins of the extracellular matrix: collagen I and IV, fibronectin, and laminin. There were no significant changes in proliferation or migration of epithelial cells induced by the lipid mediator.The results suggest PAF plays an important role in preventing corneal wound healing by affecting adhesion of epithelial cells and increasing apoptosis in stromal cells. PAF antagonists could be of therapeutic importance during prolonged ocular inflammation, helping to avoid loss of corneal transparency and visual acuity.

Sjogren’s Syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration of the salivary and lacrimal glands. The disease is most prevalent in postmenopausal women who exhibit salivary and lacrimal insufficiencies resulting in dry mouth and dry eye. Diagnosis of SS is variously dependent upon the detection of anti-Ro (SS-A), anti-La (SS-B) and antinuclear (ANA) autoantibodies, as well as rheumatoid factor. Biopsies of the salivary and lacrimal glands of SS patients typically reveal periductal and perivascular lymphocytic infiltrates that are predominated by CD4+ T and B lymphocytes1–6. Emerging theories of the pathogenesis of SS postulate the disease proceeds in stages with the first stage characterized by an intrinsic failure of the secretory epithelial cells7–12. Successive stages include the autoimmune response in the lacrimal and salivary glands initiated by the target cells themselves. The final stage is seen as one in which immune-mediated destruction of the secretory architecture of the cell, as well as of the nerve fibers innervating the gland, occurs.