Effect of citric acid conditioning on fibroblast cell density in periodontal wounds

The present experiment was undertaken to study the repopulation of curetted root surfaces by fibroblast-like cells in experimental periodontal wounds. 6 beagle dogs were used. After reflecting mucoperiosteal flaps, fenestration wounds were made through the buccal cortical plate exposing roots of maxillary canines in 6 beagle dogs. Exposed root surfaces were curetted to remove cementum and periodontal ligament. On 1 side, the exposed root surface was conditioned with citric acid (pH 1) for 3 min while the contralateral root was treated with distilled water. Millipore filters were placed over the wounds to prevent flap connective tissues from contacting the exposed root surface. Histometric analysis was made 10 days after wounding. Fibroblast-like cells were seen migrating into the fenestration space and were primarily aligned along the root surface. Cell densities measured at the borders and central part of the wound showed significantly lower values (p less than 0.01 and p less than 0.05) in acid-conditioned wounds compared to controls. No significant difference in cell density was noted between the borders and central part of the wound in the acid-conditioned group. In the controls, the cell density at the borders was significantly greater than at the center, suggesting active cell migration from the borders. The present findings suggest that citric acid conditioning of root dentin may result in a low cell density during the early stages of healing in experimental periodontal wounds.     

牙骨质基质提取物对两种细胞在牙根表面的细胞生物学研究

目的：观察牙骨质基质提取物能否促进牙周膜成纤维细胞、牙龈成纤维细胞向牙根表面移行、附着和趋向。方法：用细胞培养法和图像分析法分析细胞的附着和趋向。结果：发现牙骨质基质提取物能明显提高牙龈成纤维细胞和牙周膜成纤维细胞在牙根表面的附着，而且，随着培养时间的延长，牙骨质基质提取物促牙龈成纤维细胞和牙周膜成纤维细胞附着的功能更加显著。结论：牙骨质基质提取物可较好地促进牙龈成纤维细胞和牙周膜成纤维细胞在未脱矿的牙根表面上的附着、移行和趋向     

Cell and fiber attachment to demineralized dentin from normal root surfaces

The study assessed connective tissue and epithelial responses to dentin specimens (obtained from normal roots of human teeth) after surface demineralization. Rectangular dental specimens with opposite faces of root and pulpal dentin were prepared from beneath root surfaces covered by periodontal ligament. One-half of the specimens were treated with citric acid, pH 1, for 3 minutes, while the remainder served as untreated control specimens. Specimens were implanted vertically into incisional wounds on the dorsal surface of rats with one end of the implant protruding through the skin. Four specimens in each group were available 1, 3, 5 and 10 days after implantation. Histologic and histometric analyses included counts of adhering cells, evaluation of connective tissue fiber relationships and assessment of epithelial migration. Analyses within each group comparing root and pulpal surfaces showed no differences between any of the parameters. Comparisons between experimental and control groups showed that demineralized surfaces had a greater number of cells attached, fiber attachment occurred and epithelial downgrowth was inhibited. The fiber attachment to experimental specimens differed morphologically from fiber attachment to normal root surfaces: the number of fibers attached per unit length and the diameter of attached fibers were significantly less on experimental specimens. Demineralized specimens at 10 days had a distinct eosinophilic surface zone. Surface demineralization of dentin predisposed toward a cell and fiber attachment system which inhibited migration of epithelium.     

Biologic preparation of diseased root surfaces. An in vitro study

The study evaluated the effect of 2% sodium desoxycholate combined with whole plasma from a single donor on gingival fibroblast attachment to diseased root surfaces. Twenty extracted periodontally involved teeth were cut into halves buccolingually and sterilized by moist heat under high pressure. The diseased root surface of each control half was rubbed with a sterile cotton pellet saturated with Dulbecco's phosphate-buffered saline (PBS). The diseased root surface of each experimental half was rubbed with a sterile cotton pellet saturated with 2% sodium desoxycholate and then rubbed with a pellet soaked in human plasma. The control and experimental halves were placed in separate petri dishes, and a fibroblast cell suspension was added to each dish. The mean number of attached cells per half tooth was calculated for each group. Tooth surfaces treated with PBS (controls) showed a mean of 307 +/- 63 attached cells for 17 tooth halves; the experimental treated surfaces exhibited a mean of 650 +/- 130 attached cells for 16 halves. The difference between these numbers was statistically significant (P less than 0.01). These findings suggest that the desoxycholate/plasma combination enhanced in vitro fibroblast attachment to diseased root surfaces.     

The attachment of V79 and human periodontal ligament fibroblasts on periodontally involved root surfaces following treatment with EDTA, citric acid, or tetracycline HCL: an SEM in vitro study

OBJECTIVE: The present in vitro study has been designed to establish and compare the effects of citric acid, EDTA, and tetracycline HCl on human periodontally diseased roots on the structure, attachment, and orientation of V79 (primary Chinese hamster lung fibroblasts) cells and human periodontal ligament fibroblasts (HPDL). MATERIALS AND METHODS: Commercially available V79 cells and HPDL derived from healthy human third molars were used in this study. These fibroblasts were left in solution for seven days in order to attain confluence. Forty single-rooted teeth were obtained from patients diagnosed with periodontitis. The crown part was removed under constant irrigation and the root was split vertically into two equal halves, thus, yielding 80 specimens. Following scaling and root planing, the specimens were washed with phosphate buffered saline (PBS) and kept in 50 microg/ml gentamycin sulphate solution for 24 hours. The root pieces were then treated as follows: citric acid at pH 1, 24% EDTA, or with a 10% solution of tetracycline HCl and were then placed in V79 fibroblast cultures and HPDL cultures. The specimens were harvested after four weeks and were fixed in 2.5% glutaraldehyde in PBS before preparation for scanning electron microscopy (SEM). RESULTS: The behavior of V79 cells was similar to that of human periodontal ligament cells on root conditioned surfaces. V79 and HPDL showed a healthy morphology on root surfaces treated with citric acid and EDTA and a relatively unhealthy appearance on root surfaces treated with tetracycline HCl and distilled water (control group). CONCLUSION: The results suggest the use of citric acid and EDTA as root conditioning agents favorably affects the migration, attachment, and morphology of fibroblasts on human root surfaces, which may play a significant role in periodontal healing and regeneration.     

Cell and fiber attachment to demineralized dentin from periodontitis-affected root surfaces

This study assessed connective tissue and epithelial responses to dentin specimens obtained from periodontitis-affected roots of human teeth after surface demineralization. Rectangular dentin specimens with opposite faces of root and pulpal dentin were prepared from beneath root surfaces covered by sheets of calculus. One half of the specimens were treated with citric acid, pH 1, for 3 minutes, while the remainder served as untreated controls. Specimens were implanted vertically into incisional wounds on the dorsal surface of rats with one end of the implant protruding through the skin. Four specimens in each group were available 1, 3, 5 and 10 days after implantation. Histologic and histometric analyses included counts of adhering cells, evaluation of attached connective tissue fiber density and diameter, and assessment of epithelial migration. Analyses within each group comparing root and pulpal surfaces showed no differences between any of the parameters. Comparisons between experimental and control groups showed that demineralized surfaces had a greater number of cells attached, fiber attachment occurred and epithelial downgrowth was inhibited. Surface demineralization of dentin from periodontitis-affected roots predisposed toward a connective tissue attachment.     

Effect of autologous and allogenic platelet-rich plasma on human gingival fibroblast function

Oral Diseases (2012) 18 , 494–500 Objective: Platelet‐rich plasma (PRP) has been proposed as a method of delivering growth factors to enhance regeneration. The aim of this study was to investigate the use of autogenous and allogenic PRP and platelet‐poor plasma (PPP) on migration and proliferation of human gingival fibroblasts in vitro. Methods: Various concentrations of PRP, as well as PPP, were prepared from autologous and allogenic sources and applied to primary gingival fibroblasts. Migration was determined by assessing the fibroblast response to a concentration gradient. ³H‐thymidine incorporation and crystal violet colorimetric assays were utilized to assess DNA synthesis and proliferation. Results: Platelet‐rich plasma provides a significant migratory stimulus to gingival fibroblasts. Furthermore, the various concentrations of PRP (50%, 20% and 10%) do not promote DNA synthesis in the short term (24 h), but over the longer term (5 days) they stimulate an increase in cell proliferation. Compared with PPP, PRP was superior in terms of encouraging migration, but was inferior in terms of promoting DNA synthesis and cell proliferation. No difference was noted between the autologous and allogenic PRP preparations on cell function. Conclusion: Both PPP and PRP promote gingival fibroblast migration and proliferation in vitro, without differences between preparations obtained from autologous and allogenic sources.     

放线共生放线杆菌表面相关物质对人牙龈成纤维细胞DNA合成和细胞周期的影响

目的观察放线共生放线杆菌表面相关物质对人牙龈成纤维细胞的DNA合成和细胞周期的影响。方法应用流式细胞仪技术。结果100mg/L组明显抑制细胞DNA合成，细胞增殖指数（S+G2M）%降低（P<0.05）。结论此物质不仅抑制细胞的分裂、增殖，而且还抑制细胞的DNA合成。     

Enhanced replication of herpes simplex virus type 1 in human cells.

The effects of DNA-damaging agents on the replication of herpes simplex virus type 1 (HSV-1) were assessed in vitro. Monolayers of human lung fibroblast cell lines were exposed to DNA-damaging agents (methyl methanesulfonate [MMS], methyl methanethiosulfonate [MMTS], ultraviolet light [UV], or gamma radiation [GR]) at specific intervals, before or after inoculation with low levels of HSV-1. The ability of cell monolayers to support HSV-1 replication was measured by direct plaque assay and was compared with that of untreated control samples. In this system, monolayers of different cell lines infected with identical HSV-1 strains demonstrated dissimilar levels of recovery of the infectious virus. Exposure of DNA-repair-competent cell cultures to DNA-damaging agents produced time-dependent enhanced virus replication. Treatment with agent before virus inoculation significantly (p less than 0.025) increased the number of plaques by 10 to 68%, compared with untreated control cultures, while treatment with agent after virus adsorption significantly increased (p less than 0.025) the number of plaques by 7 to 15%. In a parallel series of experiments, cells deficient in DNA repair (xeroderma pigmentosum) failed to support enhanced virus replication. These results suggest that after exposure to DNA-damaging agents, fibroblasts competent in DNA repair amplify the replication of HSV-1, and that DNA-repair mechanisms that act on a variety of chromosomal lesions may be involved in the repair and biological activation of HSV-1 genomes.     

Effect of Essential Oil and Chlorhexidine Mouthwashes on Gingival Fibroblast Survival and Migration

Background: 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. Methods: 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. Results: 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. Conclusions: 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.     

A new 51Chromium assay for accurate measurement of cell attachment to demineralized and non-demineralized dentine in vitro

Fibroblast-like cells (RGF) derived from rat gingival explants were labelled with radioactive chromium (⁵¹Cr) and plated into Linbro 96-well microtitre plates, each well of which contained one 200 μm-thick slice of root. The root slices were removed at various times, washed, and counted in a gamma scintillation counter to determine the number of attached cells. The results obtained were expressed as counts per unit area of root, which could be converted to cell number per unit area. A linear correlation was established between the number of cells plated and the number of cells that attached in the range 0.6 to 10 × 10⁴ cells per well. Visual corroboration of this relationship was provided by examination of root slices using scanning electron microscopy.
A significant proportion of cells was found by this assay not to be removed from the slices of tooth using standard trypsinization procedures. In the case of demineralized tissue, more cells remained adherent to the root slices than were removed. Consequently, trypsinization and counting procedures as they are normally employed no longer provide an acceptable assay for cell attachment to root surfaces.
Cell attachment to root slices demineralized for 1/2 h at 37°C with ethylenediaminetetraacetic acid (EDTA) was compared with attachment to untreated roots. It was found that there was no significant difference in the number of cells attaching to undemineralized compared to demineralized roots over a 4 h period. Consequently, greater rates of migration and division as well as chemotaxis, rather than attachment, may account for the improved healing obtained with demineralized root surfaces as compared to undemineralized surfaces in experimental wounds of the periodontium.     

Genotoxicity and cytotoxicity of the epoxy resin-based root canal sealer AH plus

Previous studies with four rapid in vitro and in vivo test systems have shown that the epoxy resin-based root canal sealer AH26 may be genotoxic and cytotoxic (9). The purpose of this study was to determine the cytotoxic and genotoxic effects of the new resinous root canal sealer AH Plus by means of the growth inhibition test with primary human periodontal ligament fibroblasts and permanent 3T3 monolayers, the procaryotic umu test, the eucaryotic DNA synthesis inhibition test, and the in vivo alkaline filter elution test. In addition, Ames tests were performed with extracts from AH Plus. AH Plus caused only slight or no cellular injuries. Furthermore, no genotoxicity and mutagenicity were revealed by AH Plus. These data should be taken into consideration when deciding about a root canal sealer.     

碱性成纤维细胞生长因子对体外培养人牙髓细胞迁移和碱性磷酸酶活性的影响

目的:研究碱性成纤维细胞生长因子(basic fibroblast growth factor,bFGF)对体外培养人牙髓细胞迁移和碱性磷酸酶(ALP)活性的影响。方法:以组织块培养法体外获得人牙髓细胞,采用Transwell培养法和ALP活性检测法,观察10 ng/mL bFGF对体外培养人牙髓细胞迁移和分化能力的影响。结果:bFGF可显著诱导体外培养牙髓细胞迁移,与对照组相比差异有统计学意义(P<0.05),并能抑制细胞ALP活性(P<0.05),随着培养时间延长,该抑制作用更加显著(P<0.05)。结论:bFGF能促进牙髓细胞迁移,抑制ALP活性,在牙本质牙髓复合体修复中可能发挥重要作用。     

碱性成纤维细胞生长因子对体外培养人牙髓细胞增殖和迁移的影响

目的研究碱性成纤维细胞生长因子（basic fibroblast growth factor,bFGF）对体外培养人牙髓细胞增殖、迁移活性的影响。方法以常规组织块培养法体外获得人牙髓细胞,实验组分别加入终浓度为1.0、5.0、10.0和50.0ng/ml的bFGF,对照组仅加入等量的空白培养基,采用CCK-8法分别测定450nm下各组光吸收度A值,研究bFGF对牙髓细胞增殖活性的影响;应用Transwell小室培养法,研究bFGF对牙髓细胞迁移活性的影响。结果与对照组相比,bFGF在1.0～50.0ng/ml浓度下可促进牙髓细胞的增殖（P〈0.05）,bFGF最低显效浓度为1.0ng/ml,最佳显效浓度为10.0ng/ml。bFGF在1.0～50.0ng/ml浓度下诱导细胞迁移（P〈0.05）。结论 bFGF可诱导体外培养人牙髓细胞的增殖和迁移,在牙髓牙本质复合体修复中可能发挥重要作用。     

牙骨质基质提取物促进牙周细胞在牙根表面附着的研究

目的 明确附着的牙根表面的牙骨质基质提取物具有促进牙周细胞附着的方法。方法 采集健康的牙龋组织和牙周膜,体外培养牙龈成纤维细胞和牙周膜成纤维细胞。从因正畸需拔除的健康牙体表面获得牙骨质基质提取物,分别观察不同作用时间,不同浓度的牙骨质基质提取物对牙龈成纤维细胞和牙周膜成纤维细胞在牙体表面附着的影响,结果 牙龈成纤维细胞,牙周膜成纤维细胞均对牙骨质基质提取物有浓度和时间依赖性,最佳作用时间为２小时,     

Inhibited proliferation of human periodontal ligament cells and gingival fibroblasts by Actinobacillus actinomycetemcomitans: involvement of the cytolethal distending toxin

Actinobacillus actinomycetemcomitans can inhibit fibroblast proliferation. The objective of this study was to characterize the early proliferative responses of human periodontal ligament cells (PDLC) and gingival fibroblasts (GF) to A. actinomycetemcomitans components and to investigate the possible involvement of the cytolethal distending toxin (cdt) produced by this bacterium. The PDLC and GF were challenged with surface components of A. actinomycetemcomitans. Both DNA and protein synthesis as well as cell lysis or apoptosis were assayed for a 6-h period after addition of the bacterial extract. Unlike the controls, inhibition of DNA synthesis had already occurred in the challenged cells at the end of the initial 3- to 6-h period. No lysis or apoptosis was detected, and the total protein synthesis remained unaffected. The persistence of the effect on cell growth was confirmed after a 72-h period of challenge, during which the cells remained viable but exhibited an elongated and distended cell body. No significant differences were observed between PDLC and GF. When a cdt-knockout strain of A. actinomycetemcomitans was used almost no inhibitory effect on cell proliferation was observed. It was concluded that A. actinomycetemcomitans causes a non-lethal inhibition of proliferation in PDLC and GF as a result of an early arrest of DNA synthesis. Cytolethal distending toxin is responsible for most of this effect. This bacterial property may compromise tissue homeostasis in the periodontium.     

Cell and fiber attachment to demoralized dentin A comparison between normal and periodontitis-affected root surfaces

The purpose of the present study was to compare and contrast cellular, connective tissue, and epithelial responses to dentin specimens derived from the roots of either normal or periodontitis-affected human teeth after surface demineralization. Rectangular dentin specimens, with opposite faces of root and pulpal dentin, were derived from beneath root surfaces covered by periodontal ligament (normal) or calculus-covered areas of periodontitis-affected teeth. In each of the groups, the specimens were treated with citric acid (pH 1 for 3 min), whereupon they were implanted transcutaneously into incisional wounds on the dorsal surface of rats with one end of the implant protruding through the skin. 4 specimens were available in each group at 10 days after implantation. Histologic and histometric analyses of the root surfaces of the implants included counts of adhering cells, evaluation of connective tissue fiber relationships, and assessment of epithelial migration. New connective tissue attachment with inhibition of epithelial migration occurred in both groups. Cementum formation was not present. Comparisons between the groups showed no significant differences regarding length of implant surface adjacent to connective tissue, number of attached cells, or density and diameter of attached fibers. The fiber attachment system which had developed on these demineralized surfaces seemed intrinsic to the connective tissue location, and differed morphologically from corresponding fibers attaching the root surface in a normal periodontium. It was concluded that there were no observable differences between the new connective tissue attachment systems which developed on demineralized dentin from either normal or periodontitis-affected root surfaces.     

Effect of enamel matrix derivative on human periodontal fibroblasts: proliferation, morphology and root surface colonization. An in vitro study

BACKGROUND: Several clinical trials have shown the effectiveness of Emdogain(R) (EMD) in promoting tissue regeneration, even though the underlining biological mechanism is still poorly known. OBJECTIVES: The aim of the present study was to verify the effect of EMD on the proliferation of human periodontal ligament (PDL) fibroblasts and on their colonization and differentiation following contact with the root surface of extracted teeth in vitro. METHODS AND RESULTS: Fibroblasts from PDL were seeded on Petri dishes and cell growth was evaluated by cell counting in the presence and absence of EMD, after 1, 3 and 8 d of culture. A significant effect of EMD upon cellular proliferation at d 3 and 8 was detected. When PDL cells were grown for 12 d with EMD on etched human root surface, a change in cell morphology was observed. Scanning electron microscopy revealed that cells grown on root EMD-treated surface present a body with a flattened surface closely adherent to the substrate and an outer smooth surface rounded in shape. From the flattened surface some thin and elongated cellular processes connecting with the substrate were also observable. PDL cells grown on EMD-treated surface showed lack of alkaline phosphatase activity, as some authors noticed in cementoblasts in vitro. CONCLUSIONS: In conclusion, our data indicate that EMD enhances human PDL fibroblast proliferation. Furthermore, the cells in the presence of EMD show morphological changes that make them more similar to cementoblasts than to fibroblasts, suggesting a process of cellular differentiation that could play an important role in periodontal tissue repair.     

Proliferative effect of growth factors TGF-beta1, PDGF-BB and rhBMP-2 on human gingival fibroblasts and periodontal ligament cells

The regeneration of periodontal tissues lost due to periodontal disease requires cell migration, differentiation and proliferation. Several procedures have been proposed to promote wound healing events such as the application of growth factors including PDGF-BB, TGF-beta1 and rhBMP-2. The purpose of this study was to evaluate the mitogenic responses of human periodontal ligament cells and gingival fibroblasts to PDGF-BB, TGF-beta1 and rhBMP-2. Human periodontal ligament cells were isolated from the mid root of three maxillary third molars extracted from three adult patients with moderate periodontitis and gingival fibroblasts were obtained from two patients also affected by moderate periodontitis, who underwent periodontal surgery. Cells were grown in 24-well dishes. On day 2 of quiescence, new medium was added with PDGF-BB or TGF-beta1 or rhBMP-2 at the concentration of 10 ng/ml. To determine the effects of the test agents on cell proliferation, DNA synthesis was estimated by measuring [3H] thymidine incorporation. After 48h of incubation the cells were processed and subject to scintillation counting. Counts per minute (cpm/ well) were determined for each sample. The results of this study demonstrated that PDGF-BB acts like a strong mitogenic agent for human periodontal ligament cells and gingival fibroblasts, TGF-beta1 mostly supports the proliferation of these cells and rhBMP-2 had an opposite effect on cell mitosis.     

Cementum attachment protein enriches putative cementoblastic populations on root surfaces in vitro

We tested the capacity of cementum attachment protein (CAP) to recruit putative cementoblastic populations to root surfaces in vitro by determining the phenotypic expression of periodontal ligament cloned cell populations. The clones were derived from cells that attached to either CAP-coated (experimental) or uncoated (control) root slices. Root slices were co-cultured with primary human periodontal ligament cells. Cloned and parent populations were analyzed for their capacity to express alkaline phosphatase (AP), osteopontin, bone sialoprotein (BSP), and CAP and to form mineralized tissue in vitro. The percentage of CAP- and BSP-positive clones was significantly higher in the experimental clones than in the controls. The percentage of cells positive for AP, BSP, and CAP was higher in the experimental clones than in their control counterparts. Mineralized tissue formation was observed only in the cell populations derived from the CAP-coated root slices. These results indicate that CAP is capable of recruiting putative cementoblastic populations on root slices in vitro and therefore might play an important role in cementogenesis during periodontal homeostasis and wound healing.