Trypan blue exclusion principle in the evaluation of fibroblast attachment in vitro using V79 cells on the conditioned root surface.

OBJECTIVE: Various studies have shown that the root surface condition may play an important role in wound healing. Root surface demineralization has been shown to promote the establishment of new connective tissue attachment. Various agents, including citric acid, ethylenediaminetetraacetic acid (EDTA), and tetracycline, have been used to try to achieve a root surface that is biocompatible with the adjacent periodontal cells. Traditional in vitro studies have used periodontal ligament fibroblasts and scanning electron microscopic studies to check the efficacy of these root-conditioning agents. The objective of this study was to assess the efficacy of trypan blue to evaluate fibroblast attachment in an in vitro model using V79 fibroblasts (embryonic Chinese hamster lung fibroblasts) on root specimens treated by citric acid, EDTA, and tetracycline. METHOD AND MATERIALS: Citric acid-, EDTA-, and tetracycline-treated root specimens were placed in cultures of V79 cells and in human periodontal ligament cells, which acted as a control. The treated root specimens were removed from the cultures after 4 weeks and immediately treated with 1% trypan blue. Trypan blue was selected because it can stain only nonvital tissues. RESULTS: The root specimens placed in V79 and human periodontal ligament cultures showed unstained areas, indicating the presence of vital cells, in contrast to the stained areas, which represented the areas devoid of cells. CONCLUSION: This in vitro screening model based on the trypan blue exclusion principle may be used for immediate evaluation of the efficacy of various root-conditioning agents. V79 cells may be used as an alternative to human periodontal ligament fibroblasts in the evaluation of the efficacy of root conditioning agents.     

Behavior of human periodontal ligament cells on CO2 laser irradiated dentinal root surfaces: an in vitro study

OBJECTIVE: The aim of this study was to investigate the in vitro attachment behavior of human periodontal ligament fibroblasts on periodontally involved root surface after conditioning with CO2 laser and to compare its efficacy with chemical conditioning agents, namely tetracycline hydrochloride, citric acid, hydrogen peroxide (H2O2) and EDTA, using scanning electron microscopy. METHODS: A total of 84 scaled and root-planed specimens from periodontally involved single-rooted human teeth showing hopeless prognosis were selected and assigned to two groups. One group was lased with a CO2 laser (from 5 cm at 3 W for 0.8, 1.0 and 1.2 s), and the other group was treated with either tetracycline hydrochloride (2.5%), citric acid (saturated solution, pH 1), H2O2 (6%) or EDTA (5%; pH 7.4) for 3 min. The specimens were then seeded with human periodontal ligament fibroblasts, incubated for either 12 h or 24 h, and then the cell attachment behavior was observed. RESULTS: CO2 laser irradiation for 1.0 s was found to be the most efficient, showing consistently good cell attachment with the highest mean value (15.00 +/- 3.41 cells/10,000 microm2 after incubation for 12 h and 29.17 +/- 2.04 cells/10,000 microm2 after 24 h), followed by irradiation for 0.8 s (13.11 +/- 3.04 cells/10,000 microm2 after incubation for 12 h and 22.91 +/- 7.10 cells/10,000 microm2 after 24 h). Charring was observed following irradiation for 1.2 s. Amongst chemical conditioning agents, citric acid was found to be the most efficient, with a mean cell attachment of 17.82 +/- 2.16 cells/10,000 microm2 after incubation for 12 h and 23.62 +/- 1.94 cells/10,000 microm2 after 24 h. EDTA and H2O2 did not do well in the study. CONCLUSION: The results suggest that CO2 laser irradiation for 1.0 s may promote comparatively better attachment of periodontal ligament fibroblast on dentinal root surfaces than the conventional chemical conditioning agents used in the study.     

A study of attached and oriented human periodontal ligament cells to periodontally diseased cementum and dentin after demineralizing with neutral and low pH etching solution

BACKGROUND: This study aimed to evaluate quantitatively the migrated, attached, and oriented human periodontal ligament cells (HPLC) to periodontally diseased cementum and dentin after demineralization with low and neutral pH etching solution. METHODS: Human teeth, extracted due to periodontal diseases, were scaled and root planed so that cementum remained on one longitudinal half of the root and dentin was exposed on the other half. Forty root slices of 200 microm thickness, obtained from the mid-third of these roots, were divided into four groups: group 1, demineralized with a saturated solution of citric acid (pH 1.20); group 2, with 24% EDTA (pH 7.04); group 3, with tetracycline HCl 100 mg/ml (pH 2.00); and group 4, only scaled and root planed (control). Root slices were placed over the confluent HPLC in 35 mm culture dishes and incubated. Phase contrast microscopic photographs of attached and oriented refractile HPLC to root surfaces were taken at days 1, 3, and 7. Quantitative analysis of attached and oriented HPLC from these photographs was done using special software. RESULTS: The results showed no significant difference in the attachment and orientation index of HPLC to cementum compared to dentin in any method of demineralization at each time point (P >0.05), although there was a significantly higher cell attachment and orientation index to demineralized dentin with EDTA and citric acid than to non-etched dentin and to all 3 demineralized cementum surfaces compared to non-etched cementum (P<0.05). CONCLUSIONS: EDTA and citric acid demineralization may enhance HPLC attachment and orientation to the root surface, and it is not always necessary to remove excessive cementum when the demineralizing procedure is used.     

Attachment of human gingival fibroblasts to periodontally involved root surface following scaling and/or etching procedures: a scanning electron microscopy study

This study evaluated, in vitro, fibroblast attachment to periodontally involved root surfaces which were either root planed or acid/chelated by different agents. Specimens were divided into 3 groups of 12 specimens each. The root surfaces were root planed with a Gracey 7/8 curette, an EMS or an Amdent piezo-electric scaler and treated with saline, citric acid, tetracycline hydrochloride or EDTA to produce different surface textures. They were then cultured with fibroblasts for 72 h and examined by scanning electron microscopy. There was a significantly greater number of fibroblasts attached to specimens treated with citric acid, tetracycline and EDTA than to those root planed only. Furthermore, fibroblasts were more likely to attach to rough-surfaced than to smooth-surfaced specimens.     

贫血小板血浆和根面脱矿对人牙周膜成纤维细胞在病变牙根面附着和增殖的影响

目的：观察贫血小板血浆和根面脱矿单独及联合应用对人牙周膜成纤维细胞在病变牙根面的附着及增殖的影响,探讨PPP和根面脱矿处理在促牙周组织再生中的可能作用。方法：实验分4组,经PPP、EDTA和两者联合处理的病变根片的3组作为实验组,未处理的病变根片作对照组。分别通过细胞计数法、四唑盐比色法[MTT]和扫描电镜检测人牙周膜成纤维细胞在不同处理病变牙根表面的附着、增殖和形态。结果：与未处理组相比,PPP组及根面脱矿组均能促进人牙周膜成纤维细胞对病变根面的附着（P〈0.05）,二者联合处理促细胞附着效果明显增强（P〈0.01）;未处理组中人牙周膜成纤维细胞体外培养48h与培养24h相比,细胞在病变根片上的无增殖（P〈0.05）;单独PPP或脱矿处理组中人牙周膜成纤维细胞体外培养48h与培养24h相比,细胞在病变根片上的附着及增殖增加,但无统计学意义,PPP和根面脱矿联合处理组中细胞在病变根片上的附着及增殖明显增加（P〈0.05）。结论：PPP能牢固地附着于病变根面,PPP和根面脱矿单独和联合处理病变根片能明显加强人牙周膜成纤维细胞的附着和增殖,而PPP和根面脱矿联合处理还有显著的促细胞增值效应。     

Effects of various root surface treatments on initial clot formation: a scanning electron microscope study

A study was conducted to evaluate using SEM the earliest events of initial blood clot formation on periodontally diseased root surfaces given various treatments. Six teeth with periodontal disease were extracted from six different subjects and were studied in two individual groups. In the first group the root surfaces were divided into three individual treatment areas: (a) intact periodontal ligament, (b) planed, (c) planed and also treated with 1% citric acid. The root surfaces in the second group were likewise divided into three treatment areas: (a) intact periodontal ligament, (b) planed, (c) planed and topically conditioned with tetracycline HCl. All the roots were reinserted into the original extraction sites, and then removed at either "zero" (less than 5 s), one or 3 min and prepared for SEM evaluation. It was observed that organized clot formation occurred more rapidly in the treatment areas where both root planing and topical conditioning with tetracycline HCl had been done.     

用米诺环素处理病变牙骨质的体外效果

目的 用盐酸米诺环素处理牙周炎患牙的牙骨质，观察其表面结构和牙周膜细胞在其上附着的影响。方法 选暴露于牙周袋的根面，制备成牙骨质片，实验组用2.5%盐酸米诺环素液涂擦，对照组用生理盐水。用扫描电镜观察不同处理后，根面牙骨质的超微结构及牙周膜细胞在不同处理根面上附着生长的情况。结果 盐酸米诺环素处理后的根面，无玷污层，有胶原纤维暴露；牙周膜细胞在其上的伸展和生长优于对照组，结论 盐酸米诺环素处理可使病变牙骨质胶原纤维暴露；牙周膜细胞在其上的伸展和生长优于对照组。结论 盐酸米诺环素处理可使病变牙骨质胶原纤维暴露，促进牙周膜细胞在根面上的伸展和生长。     

Human periodontal ligament fibroblast behavior on chemically conditioned dentine: an in vitro study.

BACKGROUND: Chemical root conditioning is widely used in an attempt to improve the outcome of regenerative periodontal surgery, but its effect on connective tissue cell proliferation and biosynthetic activity has been poorly studied. The goal of the present study was to test in vitro the consequences of conditioning human dentine by citric acid or minocycline on the behavior of attached human periodontal ligament (HPDL) cells in terms of proliferation, protein synthesis and morphological appearance. METHODS: HPDL cells were seeded on powdered human dentine, either untreated or conditioned for 3 minutes with 3% citric acid or 2.5% minocycline HCI. Scanning (SEM) and transmission (TEM) electron microscopic observations were performed, and 3H-thymidine and 3H-proline incorporation tests were used to evaluate the proliferative and the biosynthetic activities. RESULTS: Cell spreading was already evident and the penetration of cytoplasmic processes into dentinal tubules were frequently observed on all dentine types after 2 hours of attachment. After 24 hours of incubation, citric acid conditioning promoted an intense spreading of the cells, while minocycline HCI conditioning induced the formation of a dense feltwork of cellular processes. HPDL fibrolasts adherent to both types of surface-conditioned dentine exhibited a significantly higher rate of proliferation (P<0.01) as well as a significantly higher level of total protein and of collagen synthesis (P<0.01) than on untreated dentine. CONCLUSIONS: These data suggest that during periodontal surgery a conditioning of the root surface by citric acid or by minocycline HCI could promote the attachment, the proliferation, and the biosynthetic activity of HDPL, prerequisites to periodontal regeneration.     

Scanning electron microscopy analysis on root surfaces before and after scalling and citric acid and EDTA conditioning: an "in vitro" study

The most benefic root surface for periodontal regeneration has not been known. Looking for this surface this study used 14 human teeth prepared as testing corpus and treated with scaling and conditioning with citric acid and EDTA, and analysed in scanning electron microscopy. The health teeth surfaces showed regular grains representing broken periodontal ligament fibers by exodontics. The contaminated surfaces showed calculus masses, very porous, and resorption areas. When scalled with hand curettes the surfaces was covered by smear layer. The citric acid conditioning was efficient on smear layer removal, as well as the EDTA gel, and the EDTA liquid solution was not efficient on smear removal.     

Fibroblastic growth and attachment on hydroxyapatite-coated titanium surfaces following the use of various detoxification modalities. Part I: Noncontaminated hydroxyapatite.

Hydroxyapatite-coated titanium alloy test strips were treated with chlorhexidine gluconate, stannous fluoride, citric acid, tetracycline HCl, polymyxin B, hydrogen peroxide, and a plastic Cavitron tip: untreated sterile strips served as controls. The strips were incubated with cultured human gingival and periodontal ligament fibroblasts. Image analysis of three photomicrographs of each test strip (original magnification x350) indicated that the tetracycline HCl treatment resulted in significantly greater cellular surface area coverage compared with the other treatments. Citric acid and the plastic Cavitron tip also stimulated cell attachment, although the results from the Cavitron tip were not significantly different from citric acid or the other treatment groups. The remainder of the modalities and the untreated cellular controls experienced similar cellular coverage.     

Ultrastructural study of initial attachment by human fibroblast-like cells on tooth roots in vitro

The purpose of this study was to determine the longitudinal effect of demineralized treatment of root surfaces on initial attachment, growth and differentiation of human periodontal ligament fibroblast-like cells in vitro. Cementum and dentin fragments were prepared from intact extracted human teeth for orthodontic reason. The root fragments of one group were not demineralized. Those of the other groups were demineralized by either citric acid (pH = 1.0, 3 min) or EDTA (pH = 7.4, 30 min). Plastic sheets served as controls. Human periodontal ligament fibroblast-like cells were incubated on root fragments and plastic sheets. After incubation, the root fragments and plastic sheets were examined by electron microscopy. The collagen fibers were exposed to the root surface by demineralized root surface. The exposed collagen fibers showed an effect on the cell attachment and growth, and the cells produced collagen fibers in the extra-cellular space of the root surface. Demineralization of dentin fragments were more strongly affected in cell attachment, growth and differentiation than demineralization of cementum fragments. Citric acid demineralization of dentin fragments had a greater effect on cell attachment, growth and differentiation than EDTA demineralization of dentin fragments. The results suggest that citric acid demineralization of dentin fragments may provide the most effective dental surface for the establishment of connective tissue attachment after periodontal treatment.     

Citric acid treatment of periodontitis-affected cementum

Previous studies have described an inconsistent histological occurrence of a zone of surface demineralization on periodontitis-affected cementum following treatment with citric acid, and a lack of connective tissue attachment to the latter surfaces. In view of these findings, the purpose of the present study was to use scanning electron microscopy to examine the surface morphology of cementum from normal and periodontitis-affected root surfaces following citric acid treatment for differences in the effects of the demineralizing solution on these surfaces. Cementum surfaces were derived from the roots of extracted human teeth from areas beneath attached periodontal ligament fibers (normal) and calculus deposits (periodontitis-affected). 5 specimens were evaluated in both groups. Periodontal ligament fibers were removed from normal root surfaces with a curette, and calculus deposits were removed from periodontitis-affected root surfaces using an ultrasonic scaler. The resultant 5 specimens in each group were then sectioned in half, one-half serving as the untreated control and the other as the experimental, citric acid treated specimen. Experimental specimens were immersed in a saturated solution of citric acid, pH 1 for 3 min and then rinsed in tap water. Both control and experimental specimens were dehydrated in ethanol, critical-point dried, sputter-coated with gold and examined in the scanning electron microscope for morphological characteristics. Citric acid treatment of cementum from normal root surfaces produced an undulating, markedly fibrillar surface morphology which is consistent with the exposure of a fibrillar, collagen substrate. Periodontitis-affected cementum, however, was not appreciably altered in appearance by the citric acid treatment, having only a faint mat-like surface texture. (ABSTRACT TRUNCATED AT 250 WORDS)     

SEM observations of the attachment of human periodontal ligament fibroblasts to non-demineralized dentin surface in vitro

PURPOSE: The purpose of this investigation was to study in-vitro the attachment behavior of human periodontal ligament (HPDL) fibroblasts to nondemineralized dentin surface using scanning electron microscope. STUDY DESIGN: Thirty root slices of freshly extracted human teeth of 4 mm thickness as well as six 5x5 mm glass slides used as a control were used in this study. The dentin surface of the root slices was not treated with any chemicals to remove the smear layer. The root slices and the glass slides were placed in tissue culture clusters and an amount of 1 ml of HPDL fibroblast cell suspension was placed over the dentin surface of the root slices and the glass slides. They were then placed into an incubator at 37 degrees C and 100% humidity for 4, 24, and 72 hours. At the end of the incubation, the cells were fixed with glutaraldehyde and examined microscopically. RESULTS: Different shapes of fully spread cells were seen. The cells were attached firmly to the dentin surface by the cytoplasmic extension of the lamellipodia and microvilli which were seen extending inside the opening of the dentinal tubules. CONCLUSION: It was concluded that the human dentin surface provided an excellent surface for attachment of periodontal ligament fibroblasts. In addition, the smear layer did not affect the cell attachment.     

The effect of local delivery of PDGF-BB on attachment of human periodontal ligament fibroblasts to periodontitis-affected root surfaces--in vitro

BACKGROUND/AIMS: The purpose of this study was to determine at what concentration does platelet-derived growth factor-BB (PDGF-BB) provide for optimal stimulation of human periodontal ligament fibroblasts (PDL) to adhere to periodontitis affected root surfaces. METHOD: 80 root dentine specimens were prepared from extracted periodontally diseased teeth obtained from patients ranging in age between 35 to 60 years. The root dentine specimens were associated with the subgingival area opposing the periodontal pocket for each extracted tooth. 10 healthy root dentine specimens were obtained from teeth extracted for orthodontic reasons and served as controls. The specimens were distributed into 9 groups (10 specimens in each). In group 1, PDL fibroblasts were cultured on the specimen surface of a diseased treated control. In group 2, PDL fibroblasts were cultured on the specimen surface of a healthy control. In groups 3 to 9, PDL fibroblasts were cultured on a pre-treated specimen surface with concentrations ranging from 5, 10, 20, 50, 100, 200 and 300 ng/ml PDGF-BB, respectively. After 24 h incubation, the media were removed, specimens were fixed, processed for SEM viewing and photographed at 750x. Fibroblast adherence was measured by counting number of cells within a standard test area and cell morphology was scored. RESULTS: Findings suggest dentine specimens pretreated with 5, 10 and 20 ng/ml PDGF-BB were not significantly different in number of adherent cells from the diseased treated control. However, at concentrations of 50, 100, 200 and 300 ng/ml, a highly significant increase in number of adherent fibroblasts was detected when compared to the diseased treated control. At these concentrations, the cell morphology was comparable to that of the healthy control. CONCLUSIONS: PDGF-BB in concentrations equal to or greater than 50 ng/ml demonstrates a significant stimulation of PDL cells adherence to periodontal diseased root surfaces. Since the higher concentrations resulted in similar effects as obtained by 50 ng/ml, it may therefore be considered that this concentration provides for optimal stimulation of human periodontal ligament fibroblasts (PDL) to adhere to periodontitis-affected root surfaces.     

A morphological comparison of radicular dentin following root planing and treatment with citric acid or tetracycline HCI

The conditioning of root surfaces with saturated solutions of citric acid or tetracycline is unpredictable in facilitating new attachment, perhaps due to the low pH of these solutions which may be denaturing the organic matrix of the root as well as demineralizing the surface. The purpose of the present study was to compare the effects of a saturated solution of citric acid (pH = 1) with that of a 0.5% solution of tetracycline HCl (pH = 3.2) on radicular dentin with regard to the removal of the smear layer, exposure of dentinal tubule openings, and demineralization of the peritubular dentin. 10 bovine incisors were used in this study. The crowns and apical 1/3 of the root were resected and the resulting root segments were then frozen in icy freon. The cementum was fractured off of the root to produce a fracture-exposed, non-instrumented dentin surface. This fracture-exposed dentin surface was divided into 4 specimens, the 1st being a fracture-exposed, non-instrumented dentin control specimen (FE). After removal of the (FE) specimen from the root segment, the remainder of the fracture-exposed dentin surface was thoroughly root planed and then subdivided into the 3 remaining specimens. One of these specimens served as the root planed dentin surface (RP); another specimen (CA) was immersed in saturated citric acid (pH = 1) for 5 min and then washed in water for 5 min; the final specimen (T) was immersed in a 0.5 mg/ml solution of tetracycline HCl for 5 min and rinsed in water for 5 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning electron microscopy study of the effect of tetracycline HCl on smear layer removal and fibrin network formation

Scanning electron microscopy (SEM) was used to evaluate root surface characteristics of human teeth affected with periodontitis following periodontal instrumentation and topical application of tetracycline HCl (TTC-HCl; pH 1.6; 4 min). Specimens were randomly assigned to periodontal instrumentation alone (control 1); periodontal instrumentation plus TTC-HCl (test 1); periodontal instrumentation plus trypsin solution after extraction (control 2); and periodontal instrumentation plus TTC-HCl plus trypsin solution after extraction (test 2). Tetracycline solution was applied with a cotton pellet. Twenty-two single root periodontitis affected human teeth scheduled for extraction were selected. Mucoperiosteal flaps were raised, root surfaces were mechanically and chemically treated, flaps were repositioned and maintained in place for 20 min. Teeth were extracted, rinsed and placed in cold phosphate buffer solution (PBS) and control 2 and test 2 groups were treated with trypsin solution. Specimens were examined using SEM. Smear layer was successfully removed, exposing dentinal tubules; however, fibrin network formation in situ was not improved by application of TTC-HCl.     

Cell and fiber attachment to demineralized cementum from normal root surfaces

The purpose of this study was to assess connective tissue and epithelial responses to cementum (from normal human root surfaces covered by periodontal ligament) after surface demineralization with citric acid. Each rectangular specimen had a face of cementum and an opposite surface composed of pulpal dentin. One half of the specimens were treated with citric acid (experimental group), 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 for examination 1, 3, 5, and 10 days after implantation. Histologic and histometric analyses of the implants included counts of adhering cells, evaluation of attached connective tissue fiber density and diameter, and assessment of epithelial migration. At day 1, a distinct lighter staining zone was present on the surface of both cementum and dentin in the experimental group which corresponded to a zone of surface demineralization produced by the acid treatment. Histometric comparisons between experimental and control groups at 10 days showed a greater number of cells attached to demineralized cementum surfaces. Also, a connective tissue fiber attachment system had developed on these experimental surfaces, but which differed morphologically from periodontal ligament fiber attachment to normal cementum. It was concluded that citric acid treatment can surface demineralize cementum from normal roots, and that the surface demineralization of this cementum facilitated a cell and fiber attachment to the cementum surface.     

Effects of citric acid and EDTA conditioning on exposed root dentin: An immunohistochemical analysis of collagen and proteoglycans

OBJECTIVE: Preservation of structural and biochemical properties of the root dentin matrix is crucial to favor healing and regenerative periodontal processes. Aim of this study was to evaluate the biochemical characteristics of collagen and chondroitin sulphate of root dentin surfaces exposed by periodontal disease after acid conditioning by means of an immunohistochemical technique. DESIGN: Human teeth scheduled for extraction due to periodontal reason were submitted to: (A) scaling and root planning; (B) ultrasonic instrumentation; (C) no instrumentation. Teeth were then exposed to: (1) 10% citric acid; (2) 17% EDTA; (3) no etching. A double immunolabeling technique was performed to identify type-I collagen and proteoglycans and analyzed under FEI-SEM. RESULTS: Use of 10% citric acid revealed intense labeling for collagen fibrils and proteoglycans; lower labeling was found after EDTA conditioning. Unetched specimens showed residual smear layer on the dentin surface resulting in no evident surface labeling. CONCLUSIONS: This study supports the hypothesis that manual or ultrasonic instrumentation alone is not able to expose the sound dentin matrix, whereas a subsequent acidic conditioning exposes collagen fibrils and associated proteoglycans. The immunohistochemical technique revealed that despite their acidity, both citric acid and EDTA were able to preserve the structural and biochemical properties of the exposed dentin matrix.