Porphyromonas gingivalis Cell Wall Components Induce Programmed Death Ligand 1 (PD-L1) Expression on Human Oral Carcinoma Cells by a Receptor-Interacting Protein Kinase 2 (RIP2)-Dependent Mechanism.

Programmed death-ligand 1 (PD-L1/B7-H1) serves as a cosignaling molecule in cell-mediated immune responses and contributes to chronicity of inflammation and the escape of tumor cells from immunosurveillance. Here, we investigated the molecular mechanisms leading to PD-L1 upregulation in human oral carcinoma cells and in primary human gingival keratinocytes in response to infection with Porphyromonas gingivalis (P. gingivalis), a keystone pathogen for the development of periodontitis. The bacterial cell wall component peptidoglycan uses bacterial outer membrane vesicles to be taken up by cells. Internalized peptidoglycan triggers cytosolic receptors to induce PD-L1 expression in a myeloid differentiation primary response 88 (Myd88)-independent and receptor-interacting serine/threonine-protein kinase 2 (RIP2)-dependent fashion. Interference with the kinase activity of RIP2 or mitogen-activated protein (MAP) kinases interferes with inducible PD-L1 expression.

Influence of retinoic acid on human gingival epithelial barriers.

BACKGROUND AND OBJECTIVES: The gingival epithelium plays an important role in the protection of oral tissues from microbial challenge. Oral keratinocytes form a barrier and show various cellular contacts, including tight junctions (TJ). To analyse the barrier function in vitro the transepithelial electrical resistance (TER) is commonly used. Retinoic acid (RA) is an important signalling molecule in most tissues, including epithelial differentiation. RA signalling is mediated through three RA receptors. The aim of the study was to investigate the influence of RA on human gingival barriers in vitro. MATERIAL AND METHODS: Immortalized human gingival keratinocytes were seeded on culture plate inserts. The effect of RA with and without infection with Porphyromonas gingivalis W83 on the barrier was analysed by TER measurements. The expression of TJ proteins was investigated by western blot. RESULTS: During differentiation, mean TER increased from 16 (1 h), 43 (4 h) to 62 (6 h) Ohm × cm2 . Addition of 15 µm RA increased TER by +19 after 1 h, +25 after 4 h and +16 Ohm × cm2 after 6 h. The pan-RA receptor inhibitor BMS 493 resulted in TER values comparable to the control. The mean established TER of the control was approximately 110 Ohm × cm2 . Addition of 15 µm RA elevated TER to 127 Ohm × cm2 after 1 h, 150 Ohm × cm2 after 4 h and 189 Ohm × cm2 after 6 h (p ≤ 0.01). RA plus infection with P. gingivalis W83 further increased the TER increasing effect but could not prevent the destruction of TER induced by bacterial infection. The protein expression of the TJ proteins claudin 4 and occludin was enhanced while ZO-1 was downregulated after 1 h of RA incubation. CONCLUSION: RA provides barrier-positive elements to the gingival epithelial cell model that is accompanied by altered expression of TJ proteins.

Peri-implant tissue destruction. The Third EAO Consensus Conference 2012.

OBJECTIVE: The task of this working group was to update the existing knowledge base regarding the prevalence of peri-implant tissue destruction, the role of occlusal overload, and the outcome of non-surgical and surgical treatment. MATERIALS AND METHODS: The literature was systematically searched and critically reviewed. Four manuscripts were presented in key areas deemed to be essential for the current understanding of the magnitude of the clinical entity peri-implantitis. The role of overload as an etiological component was reviewed. Also available data on the results from non-surgical and surgical interventions for the control of tissue destruction were presented. RESULTS: The consensus statements following plenary session approval, clinical implications, and directions for future research based on the group discussions are presented in this article. The results and conclusions of the systematic review process are presented by the respective authors in the subsequent papers.

Production of interleukin-13 is influenced by the interleukin-4 -34TT and -590TT genotype in patients with aggressive periodontitis.

Aggressive periodontitis (AgP) is a specific form of periodontal disease, with rapid destruction of the tissues supporting the teeth in otherwise young healthy individuals. We recently showed a higher frequency of the interleukin-4 (IL-4) -34TT and -590TT genotype in AgP patients compared to controls (P<0.05). Herein, we demonstrated that this specific IL-4 genotype exerts its function by increasing expression of IL-4 and STAT6, and producing higher concentrations of IL-4 in activated CD4+ cells of patients with AgP. In the present study, we investigated the effects of the IL-4-specific genotype on IL-13, IL-2 and IFN-γ expression and production in activated CD4+ cells of patients with AgP and healthy controls. Results revealed higher IFN-γ and IL-2 expression and significantly increased IL-13 production in the cells of the patients who were homozygous for the -34T and -590T alleles in comparison with the patients who were homozygous for the -34C and -590C alleles (P<0.05). Results of controls with the -34C and -590C alleles were similar to those of AgP with the same genotype. To our knowledge, the present study is the first to show an effect of the -34TT and -590TT genotype on IL-13 production. There is an increased production of IL-13 by the T cells of aggressive periodontitis patients with the IL-4 genotype.

Oral mucosa model based on a collagen-elastin matrix.

BACKGROUND AND OBJECTIVE: The collagen-elastin matrix (Matriderm(®)) is used to treat deep and full-thickness burns and was recently described as a suitable scaffold for tissue engineering. The aim of the present study was to investigate the biocompatibility of Matriderm(®) for gingival use through creation of an oral mucosa model ex vivo. MATERIAL AND METHODS: Gingival fibroblasts and keratinocytes were cultured. A dermal area on the base of the collagen-elastin matrix was repopulated with fibroblasts. After 14 days, keratinocytes were seeded on this dermal area to engineer a multilayered mucosa. Analysis of the architecture was performed using light and electron microscopy. Immunohistochemical detection of collagen IV and cytokeratin was carried out. RESULTS: Based on this scaffold we generated a multilayered oral mucosa-like structure. Histological, immunohistochemical and electron microscopic analysis of the dermal/epidermal junction showed a typical basement membrane and hemidesmosomal structures. Neighboring keratinocytes formed desmosomes in the epidermal sections. Cytokeratin was detectable in all epidermal layers. These experiments revealed that the collagen-elastin matrix was highly biocompatible with gingival cells under ex vivo conditions. CONCLUSION: Employing tissue-engineering techniques with dermal and epidermal cells from the gingiva, a multilayered oral mucosa was generated and characterized with respect to biocompatibility for Matriderm(®). The results indicate that Matriderm(®) is suitable for the ex vivo growth of gingival tissue cells and is a useful scaffold with possible applications in periodontal therapy.

[Diabetes mellitus and periodontitis. Bidirectional relationship and clinical implications. A consensus document]. / Diabetes mellitus und Parodontitis. Wechselbeziehung und klinische Implikationen. Ein Konsensuspapier.

Diabetes and periodontitis are chronic diseases with an increasing prevalence in the German population. There is a bi-directional relationship between both diseases. Diabetes promotes the occurrence, the progression and the severity of periodontitis. Periodontitis complicates the glycemic control of diabetes, increases the risk of diabetes-associated complications and possibly even of its onset. In view of the existing evidence, that is still not sufficiently communicated within the medical community, an expert panel consisting of four diabetologists and four periodontists has addressed the following questions: What is the effect of diabetes mellitus on periodontitis and on periodontal therapy? What is the effect of periodontitis on diabetes mellitus? What are the practical consequences, that result for interdisciplinary treatment strategies? The treatment of periodontal infections should become an integral part of the management of diabetes, whereas glycemic control is a prerequisite for successful periodontal therapy.

Generation and functional characterization of recombinant Porphyromonas gingivalis W83 FimA.

Porphyromonas gingivalis (P. gingivalis) is regarded as a keystone pathogen in destructive periodontal diseases. It expresses a variety of virulence factors, amongst them fimbriae that are involved in colonization, invasion, establishment and persistence of the bacteria inside the host cells. The fimbriae also were demonstrated to affect the host immune-response mechanisms. The major fimbriae are able to bind specifically to different host cells, amongst them peripheral blood monocytes. The interaction of these cells with fimbriae induces release of cytokines such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor-α (TNF-α). The aim of this study was to generate recombinant major FimA protein from P. gingivalis W83 fimbriae and to prove its biological activity. FimA of P. gingivalis W83 was amplified from chromosomal DNA, cloned in a vector and transferred into Listeria innocua. (L. innocua).The expressed protein was harvested and purified using FPLC via a His trap HP column. The identity and purity was demonstrated by gel-electrophoresis and mass-spectrometry. The biological activity was assessed by stimulation of human oral epithelial cells and peripheral blood monocytes with the protein and afterwards cytokines in the supernatants were quantified by enzyme linked immunosorbent assay (ELISA) and cytometric bead array. Recombinant FimA could successfully be generated and purified. Gel-electrophoresis and mass-spectrometry confirmed that the detected sequences are identical with FimA. Stimulation of human monocytes induced the release of high concentrations of IL-1ß, IL-6, IL-10 and TNF-α by these cells. In conclusion, a recombinant FimA protein was established and its biological activity was proven. This protein may serve as a promising agent for further investigation of its role in periodontitis and possible new therapeutic approaches.

Scope, competences, learning outcomes and methods of periodontal education within the undergraduate dental curriculum: a consensus report of the 1st European Workshop on Periodontal Education--position paper 2 and consensus view 2.

The explosion in new knowledge in the last three decades has imposed important challenges in the training of all health workers, including dentists. These breakthroughs do not rapidly permeate educational cirricula. This is particularly relevant in Periodontology, because of recent breakthroughs in diagnostics and therapeutic approaches and by the advent of new knowledge on the implications of periodontal and peri-implant infections on systemic health and on ageing populations. Periodontology as one of the major oral health sciences requires a broad understanding of a spectrum of healthcare and basic sciences, together with specific education. In preparation for graduation, students must demonstrate a variety of acquired learning out-comes, which, in turn demand variety in learning and teaching methods. Based on the ADEE principles for dental education and in full respect of the Bolonga process, this paper describes the competencies and learning outcomes which are requested in periodontology for the dentist at graduation.

Use of platelet-rich plasma in periodontal surgery--a prospective randomised double blind clinical trial.

The aim of this prospective controlled randomized clinical trial was to evaluate the additional effect of platelet-rich plasma (PRP) in attachment gain. Twenty-two patients showing contralateral intrabony defects were included. Defects were randomized to beta-TCP (Cerasorb) in combination with PRP (test) or alone (control). Probing pocket depth (PPD), clinical attachment level (CAL), and relative AL (RAL) were assessed at the first, initial, re-evaluation (or basis examinations) and 6 months after surgery. Defect dimensions were recorded at baseline surgery (day 0) and during re-entry surgery (after 6 months), with vertical depth of the defect as primary outcome variable. An early healing index (EHI) was assessed 3 days, 1, 2 and 4 weeks after surgery. Both treatments led to clinical improvements. The median reduction of open vertical depth was 1.9 mm (interquartile intervals, 0.75 and 2.5 mm) at test sites, compared with 2.6 mm (1.8 and 3.5 mm) at control sites (p = 0.19, Wilcoxon). The median reductions of PPD and CAL at the four sites in close proximity to the defect in the interproximal area at test sites were 0.8 and 0.28 mm, and at control sites 0.4 and 0.13 mm, respectively. The EHI showed a reduction from grade 3 after 3 days to grade 1 after 4 weeks. PRP did not improve the results achieved with beta-TCP in the treatment of intrabony defects.

Effect of Periodontal Treatment on HbA1c among Patients with Prediabetes.

Evidence is limited regarding whether periodontal treatment improves hemoglobin A1c (HbA1c) among people with prediabetes and periodontal disease, and it is unknown whether improvement of metabolic status persists >3 mo. In an exploratory post hoc analysis of the multicenter randomized controlled trial "Antibiotika und Parodontitis" (Antibiotics and Periodontitis)-a prospective, stratified, double-blind study-we assessed whether nonsurgical periodontal treatment with or without an adjunctive systemic antibiotic treatment affects HbA1c and high-sensitivity C-reactive protein (hsCRP) levels among periodontitis patients with normal HbA1c (≤5.7%, n = 218), prediabetes (5.7% < HbA1c < 6.5%, n = 101), or unknown diabetes (HbA1c ≥ 6.5%, n = 8) over a period of 27.5 mo. Nonsurgical periodontal treatment reduced mean pocket probing depth by >1 mm in both groups. In the normal HbA1c group, HbA1c values remained unchanged at 5.0% (95% CI, 4.9% to 6.1%) during the observation period. Among periodontitis patients with prediabetes, HbA1c decreased from 5.9% (95% CI, 5.9% to 6.0%) to 5.4% (95% CI, 5.3% to 5.5%) at 15.5 mo and increased to 5.6% (95% CI, 5.4% to 5.7%) after 27.5 mo. At 27.5 mo, 46% of periodontitis patients with prediabetes had normal HbA1c levels, whereas 47.9% remained unchanged and 6.3% progressed to diabetes. Median hsCRP values were reduced in the normal HbA1c and prediabetes groups from 1.2 and 1.4 mg/L to 0.7 and 0.7 mg/L, respectively. Nonsurgical periodontal treatment may improve blood glucose values among periodontitis patients with prediabetes (ClinicalTrials.gov NCT00707369).

Establishment and characterization of immortalized human gingival keratinocyte cell lines.

BACKGROUND AND OBJECTIVE: Primary human keratinocytes are used to analyze the properties of the oral epithelium and the early stages of oral bacterial infections. In vitro, these cells are characterized by their short life span and restricted availability. Approaches for culturing these cells will end after approximately 6-10 passages as a result of entry into apoptosis. For this reason, it is important to generate cell lines suitable for obtaining an unlimited source of cells. Therefore, the aim of the present study was to generate gingival keratinocyte cell lines and to compare their in vitro behaviour with those of primary human gingival keratinocytes. MATERIAL AND METHODS: Primary human gingival keratinocytes were immortalized with a combination of the human papilloma virus onkoproteins E6 and E7. The pattern of the cytokeratins, involucrin and filaggrin was investigated by intracellular staining using flow cytometry. This method allows quantitative analysis of the expression of a variety of intracellular or extracellular markers. RESULTS: The immortalized cell lines showed many morphological similarities, expressing a cytokeratin pattern that is comparable with that of primary gingival keratinocytes. Furthermore, they developed transepithelial electrical resistance, which is a marker for the generation of tight junctions. These results indicate that the cells might be able to act as an epithelial barrier, reflecting the reaction of primary human cells. CONCLUSION: The establishment of a continuous line of human gingival epithelial cells with functional characteristics of the epithelial barrier provides a valuable in vitro model for using to study the early steps of gingival/periodontal infections.

Clinical outcomes after treatment of intra-bony defects with an EMD/synthetic bone graft or EMD alone: a multicentre randomized-controlled clinical trial.

OBJECTIVES: Comparison of the outcomes of a combination of an enamel matrix derivative and a synthetic bone graft (EMD/SBC) with EMD alone in wide intra-bony defects. MATERIAL AND METHODS: Seventy-three patients with chronic periodontitis were recruited in five centres in Germany. All patients had one wide intra-bony defect of >/=4 mm. Surgical procedures involved microsurgical technique and the modified papilla preservation flap. After debridement, defects were randomly assigned to EMD/SBC (test) or EMD (control). Assessments at baseline and after 6 months included bone sounding, attachment levels, probing pocket depths, bleeding on probing and recessions. Early wound-healing, adverse effects and patients' perceptions were also recorded. RESULTS: Both treatment modalities led to significant clinical improvements. Change in bone fill 6 months after surgery was 2.0 mm (+/- 2.1) in the test group and 2.1 mm (+/- 1.2) in the control group. A gain in clinical attachment of 1.3 mm (+/- 1.8) in the test group and 1.8 mm (+/- 1.6) in the control group was observed. One week after surgery, primary closure was maintained in 95% of the test sites and 100% of the control sites. No differences in patients' perceptions were found. CONCLUSION: The results of the present study showed similar clinical outcomes following both treatment modalities.

Induction of B7-H1 receptor by bacterial cells fractions of Porphyromonas gingivalis on human oral epithelial cells: B7-H1 induction by Porphyromonas gingivalis fractions.

The immune-regulatory B7-H1 receptor, also known as programmed death-ligand 1 (PD-L1), plays an important role in cell-mediated immune response. It is a co-signaling molecule that mediates regulation of T cell activation and tolerance and is able to negatively regulate activated T cell functions and survival. High expression of B7-H1 in host cells may contribute to the chronicity of inflammatory disorders and represents a possible mechanism of immune evasion. Porphyromonas gingivalis is regarded as a keystone pathogen in periodontitis and is able to invade host cells and disposes a variety of virulence factors including lipopolysaccharide (LPS), fimbriae and proteases such as gingipains. Based on previous studies that demonstrated the capability of P. gingivalis to induce up-regulation of PD-L1 in malignant and non-malignant oral epithelial cells, the aim of the present work was to analyse the potential of various cellular components of P. gingivalis to induce the PD-L1 receptor. Human squamous carcinoma cells and primary gingival keratinocytes were stimulated with total, inner and outer membrane fractions, cytosolic proteins, as well as LPS and peptidoglycans. PD-L1 protein expression was investigated by Western blot analysis and RT-PCR. It was demonstrated that the total membrane fraction induced the highest up-regulation in B7-H1 expression, followed by the outer and inner membrane, whereas cytosolic proteins and LPS did not. In conclusion, we provide evidence that the membrane fraction of P. gingivalis is responsible for up-regulation of the immune-regulatory receptor PD-L1 in squamous carcinoma cells and gingival keratinocytes, and thus may support immune evasion of oral carcinomas.

Oral Squamous Carcinoma Cells Express B7-H1 and B7-DC Receptors in Vivo.

B7-H1 and B7-DC ligands are members of the B7 family with important regulatory functions in cell-mediated immune response. Both receptors are ligands of the programmed death receptor PD-1. B7-H1 expression has been detected in the majority of human carcinomas in vivo. B7-H1 mediated signals are able to negatively regulate activated T cell functions and survival, and enable tumor cells to overcome host response. The aim of this study was to investigate the expression of B7-H1 and B7-DC proteins in oral squamous cell carcinomas (OSCC) in vivo. Tissues from 15 samples were cryo-sected and following histological routine staining (HE), incubated with antibodies against human B7-H1 and B7-DC. Immuno-staining of pan-cytokeratin was performed to ascertain the epithelial origin of the tissue and CK 19 to demonstrate the proliferating stage. Confocal laser scanning microscopy confirmed the presence of both B7-H1 and B7-DC in all 15 OSCC. The B7-H1 and B7-DC staining was located in areas of the tissue that were identified as cancerous lesions in the previously stained HE sections before. Staining with Pan-CK and CK19 provided evidence for the epithelial origin and the proliferating stage of the tissue. The in vivo expression of the B7-H1 and B7-DC receptors in oral squamous cell carcinomas suggest that general mechanisms for immune evasion of tumors are also found in OSCC.

Chemotactic activity of substances derived from antibody-loaded tumor cells on granulocytes.

Chemotactic activity of granulocytes attracted by tumor cells loaded either with anti-ganglioside monoclonal antibodies (mAb) or with antibody-glucose oxidase conjugates (mAb-GO) was investigated. The melanoma cell line SK-Mel-28 which expresses the ganglioside GD3 at high density as well as the neuroectodermal cell line SK-N-LO which expresses GD2 were used for the experiments. In the presence of 50% human AB-serum, antibody-loaded tumor cells induced chemotactic activity on granulocytes, probably due to the generation of C3a/C5a which could be detected in serum incubated with anti-GD3 loaded SK-Mel-28 cells. Both compounds could also be detected in vivo in the plasma of patients suffering from neuroblastoma during therapy with anti-GD2 antibodies. In another set of experiments mAb-GO conjugates generating high amounts of H2O2 in the presence of glucose were bound to these tumor cells. A significant lipid peroxidation could be observed in the simultaneous presence of iron and ascorbate. The lipid peroxidation products were measured as thiobarbituric acid-reactive substances (TBARS) and were also shown to induce chemotactic effects on granulocytes.

Influence of the surface structure of titanium materials on the adhesion of fibroblasts.

Of utmost importance for the successful use of an implant is a good adhesion of the surrounding tissue to the biomaterial. In addition to the surface composition of the implant, the surface topography also influences the properties of the adherent cells. The aim of this investigation was thus to study the influence of the surface structure of the substrate on the formation of focal contacts and on the orientation of cultivated gingival fibroblasts by means of fluorescence microscopy. A further goal was to determine the effect of the material composition on the cell shape, on the assumption that in each case a lengthening of the cells can be expected to provide a more favourable adhesion behaviour than a spherical cell shape. In order to describe the shape of the cell, a shape factor was defined which was calculated from the area covered by the cells and from their circumference. To determine the influence of the surface structure, substrate platelets of cp-titanium, TiAl6V4 and TiTa30 were ground. Onto these specimens human gingival fibroblasts of the 5th to 7th passages were cultivated. After a culture time of two days the cells were fixed and stained. The number of orientated cells was determined as a function of the surface roughness of the substrate. The number of orientated cells was shown to increase---independent of the material---with increasing roughness of the ground substrate. On a polished surface the number of orientated cells was 11% (average peak-to-valley height 0.04 microns); at a peak-to-valley height of 1.36 microns the number of orientated cells increased to 72%. It could be observed that the orientated cells had a higher density of focal contacts where they were in contact with the edges of the grinding grooves. In order to determine the effect of the surface composition, gingival fibroblasts were cultured for 14 d on polished substrate specimens of cp-titanium, TiAl6V4 and TiTa30 and examined for differences in shape. The cells grown on cp-titanium and on TiTa30 had shape factors of 1.76 and 1.58 respectively, whereas those grown on TiAl6V4 had a shape factor of 0.93. The unfavourable spherical shape of the fibroblasts (resulting in a poor adhesion) grown on TiAl6V4 after a culture period of 14 d may be the result of a locally increased vanadium concentration in the substrate, with an accompanying increase in the release of toxic vanadium ions.

Surface roughness, porosity, and texture as modifiers of cellular adhesion.

Substrate topography in the micrometer range is reviewed as a modifier of the response of cultured cells and of biocompatibility when implanted into tissues. Characterization methods for substrate topography are discussed, including scanning electron microscopy, profilometry, laser scanning, and confocal microscopy. Because of the current technical limitations in reproducing micron-level topographic details, only one method, ion-beam etching, has been found suitable for texturing substrates on nonplanar surfaces.

Interleukin-1 beta concentration of gingival crevicular fluid.

The concentration of interleukin-1 beta is elevated in inflamed gingival tissue. Therefore a method for the measurement of interleukin-1 beta (Il-1 beta) in gingival crevicular fluid (GCF) using a commercially available Il-1 beta ELISA was developed. GCF was collected with periopaper strips and 4 protocols of sampling using filter paper strips were tested; the method with a recovery rate of 111.9% (SD: +/- 14.5%) was chosen for subsequent analysis of all samples. Il-1 beta concentration in GCF of periodontitis patients and a healthy control group was determined. Patients (n = 19, mean age: 29.3 years) had not been treated. The healthy control group (n = 14, mean age: 22.8 years) showed, after a hygiene regimen of 2 weeks, no clinical signs of gingival/periodontal inflammation. Probing depth, clinical attachment level, bleeding upon probing, and a modified plaque index were recorded. Il-1 beta could be detected in all GCF samples. The concentration ranged between 22.8 ng/ml and 150 ng/ml in the healthy control group and between 85.8 ng/ml and 882.2 ng/ml in the periodontitis patients. No sex-related differences were noted. According to our present results the determination of GCF Il-1 beta concentration is possible using commercially available test kits if the principle of sample preparation is adapted to the specific requirements of GCF analysis.

Effects of various hygiene procedures on the surface characteristics of titanium abutments.

The use of cleaning instruments on titanium implants may cause undesired surface alterations. In a qualitative and quantitative assessment of these alterations, 5 titanium implant abutments were treated with a steel curet, a prototype pure titanium curet, an air abrasive polishing system, and an ultrasonic system. Custom-made polymer templates, used to secure the curet to a vertical guide bar and a spring scale to maintain a constant instrument pressure, guaranteed a standardized procedure and reproducible results. The ultrasonic and the air abrasive polishing method were also standardized. Evaluation by scanning electron microscopy (SEM) revealed surface alterations for all instruments and systems except the plastic curet, which did not roughen the surface at all. The confocal laser-scanning microscope allows a 3-dimensional reproduction of these surface alterations and their direct measurement. The profilometric tracing was not sensitive enough to register the minor effects caused by the titanium curet and the air abrasive polishing system. Dimensions of the resulting surface microstructure could be determined with the laser-scanning microscope. Since the influence of such surface defects on the peri-implant tissue reaction is unpredictable, the titanium curet and the air abrasive system can only be recommended with restrictions. The steel curet and the ultrasonic system proved to be totally unsuitable for cleaning titanium implants.

Assay of ascorbic acid in human crevicular fluid from clinically healthy gingival sites by high-performance liquid chromatography.

The volume of fluid on filter paper strips was measured with a Periotron, eluted, and the ascorbic acid measured by chromatography. In preliminary experiments, pre-impregnation of the strips with citric acid increased the recovery of standard ascorbic acid from 37 to 89% and significantly reduced loss of water from the strips over a 3 min period. Samples of crevicular fluid were then collected from clinically healthy gingival sites of 21 healthy volunteers using pre-impregnated strips and assayed for ascorbic acid concentration, together with samples of blood plasma. The mean ascorbic acid concentration in gingival crevicular fluid (207.3 mumol/l; SD: +/- 81.8) was significantly higher (p less than 0.001) than the corresponding plasma concentration (mean 72 mumol/l; SD: +/- 23.3).