bluuebunny/crossref_metadata_2025 huggingface dataset:
Nanocrystalline ZnO Thin Film Synthesis Using Glycerol in Aqueous Polymeric Precursor Processing
Authors: Choppali Uma , Gorman Brian P.
Published: July 2008

Synthesis of high‐quality ZnO thin films via simple and cost effective processing technique is a major challenge. In this work, the preparation of nanocrystalline ZnO thin films by a novel polymeric precursor processing using glycerol as chelating agent is presented. The process has advantages of being cost‐effective and environment friendly. ZnO thin films were prepared by a single spin‐coating deposition of aqueous polymeric precursor prepared with zinc nitrate [Zn(NO3)2] and glycerol as chelating agent. The thermal decomposition of polymeric precursors was studied by thermogravimetric analysis and Fourier transform infrared (FTIR) spectroscopy. Annealing of these films were performed over the range of 300°–600°C, and the effect of annealing on the degree of crystallization, surface morphology, crystallite size, and optical properties was investigated. X‐ray diffraction analysis shows that the thin films are polycrystalline with wurtzite structure. The thin films are 80% dense, have crack free microstructure, and transparency of >85% in the visible region. These films exhibit absorption edge at 375 nm. On measuring at room temperature, the optical band gap energy of ZnO thin films, annealed at 450° and 600°C, was determined to be 3.295 and 3.267 eV. Room temperature photoluminescence spectra of these films show strong UV emission and a broad yellow‐green emission in the range 525–600 nm. The intensity of UV emission peak increases with increase in annealing temperature that is attributed to an improvement in crystallinity.
Detection of Pretreated Fingerprint Fluorescence Using an LED‐based Excitation System
Authors: Takatsu Masahisa , Shimoda Osamu , Onishi Kyoko , Onishi Akira , Oguri Naoki
Published: July 2008

Abstract: Optimization of a light emitting diode (LED)‐based excitation system for the detection of pretreated fingerprint fluorescence is described. Fluorescent ridges can usually be excited by irradiation with forensic light sources such as xenon arc lamps or quartz‐halogen lamps with high‐power output and suitable filters. However, they are too expensive for many crime laboratories in smaller organizations. We concentrated on LEDs which have advantages over conventional light sources in that they are simpler and of lower cost, but the power output and quality of each individual LED unit is not sufficient for the detection of weak fluorescent ridges. To resolve this subject, blue and green LED arrays composed of ninety LED units were adopted and suitable low pass filters for them were designed. An experimental system, consisting of blue and green LED arrays with the suitable low pass filters for illumination, high pass filters for viewing, a digital camera and a computer, was tested. The fluorescent images of cyanoacrylate ester fumed/rhodamine 6G stained fingerprint on white polyethylene sheet and weak fluorescent ridges of ninhydrin/indium chloride treated fingerprint on white paper were successfully detected and photographed. It was shown that the improvement of LED beam in intensity and quality can compensate the disadvantages, resulting in well‐contrasted images.
Evaluation and Quantification of Nuclear DNA from Human Telogen Hairs*
Authors: Opel Kerry L. , Fleishaker Erica L. , Nicklas Janice A. , Buel Eric , McCord Bruce R.
Published: July 2008

Abstract:  Nuclear DNA was extracted from human telogen hairs from 60 individuals. Six to nine hairs from each individual were individually extracted. The amount of DNA recovered from each individual varied greatly, and most samples yielded a quantity of 550 pg or less per hair. A selective extraction buffer was used to remove epithelial cell DNA and the amount of exogenous DNA was determined. DNA was also quantified by real time PCR using three different sized amplicons targeting an Alu sequence. The results were used to determine the state of degradation of the extracted DNA. Different quantities of sample (<100pg, 100–500pg, >500pg) were amplified with the Miniplex kits to determine the minimum DNA template required for successful amplification. DNA recovered from hair showed degradation; however, partial profiles were obtained for those samples containing at least 60 pg using MiniSTRs.
Death Caused by a Signal Rocket—Not an Isolated Case*
Authors: Alempijević Djordje , Ječmenica Dragan , Pavlekić Snežana , Savić Slobodan , Kovačević Slobodan
Published: July 2008

Abstract:  Fatalities related to sporting events are predominantly caused by blunt force injuries especially due to the emotional involvement of crowd, but occasionally other types of trauma are reported as well. A case of very rare trauma caused by shooting with a hand‐held parachute signal rocket during a football match is presented. A 17‐year‐old football fan sustained fatal injuries, a combination of mechanical trauma caused by rocket penetration, as well as extensive thermal burning of the thoracic viscera. Analysis of the event was based on autopsy findings and evidence produced by medicolegal and ballistic experts. Improper use of a hand‐held signal rocket, designed for marine distress signals, may cause serious injuries either mechanically, due to explosion, or as a result of thermal discharge. In the reported case, pattern of injuries is discussed, and medical finding corroborated to other available evidence. The presented case is a reminder that the forensic pathologist should be informed accordingly on the type and features of weapon suspected to produce injury, to be able to understand traumatic changes, and look for potential presence of foreign bodies at postmortem examination.
Developmental Validation of a Cannabis sativa STR Multiplex System for Forensic Analysis
Authors: Howard Christopher , Gilmore Simon , Robertson James , Peakall Rod
Published: July 2008

Abstract:  A developmental validation study based on recommendations of the Scientific Working Group on DNA Analysis Methods (SWGDAM) was conducted on a multiplex system of 10 Cannabis sativa short tandem repeat loci. Amplification of the loci in four multiplex reactions was tested across DNA from dried root, stem, and leaf sources, and DNA from fresh, frozen, and dried leaf tissue with a template DNA range of 10.0–0.01 ng. The loci were amplified and scored consistently for all DNA sources when DNA template was in the range of 10.0–1.0 ng. Some allelic dropout and PCR failure occurred in reactions with lower template DNA amounts. Overall, amplification was best using 10.0 ng of template DNA from dried leaf tissue indicating that this is the optimal source material. Cross species amplification was observed in Humulus lupulus for three loci but there was no allelic overlap. This is the first study following SWGDAM validation guidelines to validate short tandem repeat markers for forensic use in plants.
Ossification of Laryngeal Structures as Indicators of Age*
Authors: Garvin Heather M.
Published: July 2008

Abstract: As the role of forensic anthropologists expands to the medical examiner setting, their expertise is being applied beyond the traditional dry skeletal material. In such scenarios radiographic techniques can be applied when maceration is not appropriate. This study explores the use of radiographic analysis of laryngeal structures for age‐at‐death determination. Isolated human laryngeal structures (n = 104) from individuals between the ages of 15 and 89 were removed at autopsy and radiographically examined. The cricoid and individual regions of the thyroid cartilage were scored according to degree of ossification, and the relationship between age and degree of ossification statistically examined. A previously published study on age‐determination from thyroid ossification by Černý was assessed for accuracy. The results of the study indicate that although a consistent sequence in the ossification of laryngeal structures exists, variations in timing does not permit narrow age range estimates. Consequently the method presented by Černý is inaccurate and should not be used in the forensic setting.
An Insight into Forensic Document Examiner Expertise for Discriminating Between Forged and Disguised Signatures
Authors: Dyer Adrian G. , Found Bryan , Rogers Doug
Published: July 2008

Abstract:  It has previously been shown that forensic document examiners (FDEs) have expertise in providing opinions about whether questioned signatures are genuine or simulated. This study extends the exploration of FDE expertise by evaluating the performance of eight FDEs and 12 control subjects at identifying signatures as either forgeries or the disguised writing of a specimen provider. Subject eye movements and response times were recorded with a Tobii 1750 eye tracker during the signature evaluations. Using a penalty scoring system, FDEs performed significantly better than control subjects (t = 2.465, p = 0.024), with one FDE able to correctly call 13 of the 16 test stimuli (and three inconclusive calls). An analysis of eye movement search patterns by the subjects indicated that a very similar search strategy was employed by both groups, suggesting that visual inspection of signatures is mediated by a bottom up search strategy. However, FDEs spent greater than 50% longer to make a decision than the control group. The findings are suggestive that for some stimuli FDEs can discriminate between forgeries and disguises, and that this ability is due to a careful inspection and consideration of multiple features within a signature.
Unusual Suicide with a Chainsaw
Authors: Tournel Gilles , Dédouit Fabrice , Balgairies Axelle , Houssaye Cédric , De Angeli Bérengère , Bécart‐Robert Anne , Pety Nicolas , Hédouin Valéry , Gosset Didier
Published: July 2008

Abstract:  Described here is a case of suicide with the use of a chainsaw. A female suffering from schizophrenia committed suicide by an ingenious use of a chainsaw that resulted in the transection of her cervical spine and spinal cord. The findings of the resulting investigation are described and the mechanism of suicides with the use of a chainsaw is reviewed. A dry bone study was realized to determine the bone sections, the correlation between anatomic lesions and characteristics of chainsaw. The damage of organs and soft tissues is compared according to the kinds of chainsaw used.
An Examination of the Sequence of Intersecting Lines Using Attenuated Total Reflectance–Fourier Transform Infrared Spectral Imaging*
Authors: Bojko Katherine , Roux Claude , Reedy Brian J.
Published: July 2008

Abstract: In this study, the potential of attenuated total reflectance–Fourier transform infrared (ATR‐FTIR) spectral imaging as a technique to determine the sequence of line crossings was examined. The technique was successful in determining the sequence of heterogeneous line intersections produced using ballpoint pens and laser printers. By imaging at characteristic frequencies, it was possible to form spectral images showing the spatial distribution of the materials. By examining the spectral images from the inks, it was possible to determine whether the ink was above or below the toner. In blind testing, ATR‐FTIR spectral imaging results were directly compared to those obtained by eight experienced forensic document examiners using methods regularly employed in casework. ATR‐FTIR spectral imaging was shown to achieve a 100% success rate in the blind tests, whereas some incorrect sequence determinations were made by the forensic document examiners when using traditional techniques. The technique was unable to image ink‐jet printing, gel pens, roller ball pens, and felt‐tip pens, and was also unable to determine the sequence of intersecting ballpoint pen lines.
Shopping Cart Injuries, Entrapment, and Childhood Fatality
Authors: Jensen Lisbeth , Charlwood Cheryl , Byard Roger W.
Published: July 2008

Abstract:  Shopping carts may be associated with a variety of injuries, particularly in toddlers and young children. These usually relate to falls from carts or to tip‐overs. Injuries that are sustained include hematomas/contusions, abrasions, lacerations, fractures, and fingertip amputations. Fatal episodes are uncommon and are usually due to blunt craniocerebral trauma from falls. A case involving a 19‐month‐old girl is reported who became entrapped when she inserted her head through the side frame of a cart that had been removed from a supermarket and left at her home address. Death was caused by neck compression. Although rare, the potential for lethal entrapment during unsupervised play means that the presence of stray shopping carts at private residences and in public places, including playgrounds and parks, is of concern. Strategies, such as coin deposits, should be encouraged to assist in the return of such carts to supermarkets.
Investigation into “Normal” Background DNA on Adult Necks: Implications for DNA Profiling of Manual Strangulation Victims
Authors: Graham Eleanor Alison May , Rutty Guy Nathan
Published: July 2008

Abstract:  Others have investigated the role that DNA profiling could play as a method for identifying the perpetrator of manual strangulation. These studies have demonstrated that it is possible to collect offender DNA from the skin surface of a victim following physical contact. It is not known whether nonself biological material is normally present on the skin surface due to adventitious transfer occurring during innocent everyday interactions. To test the hypothesis that detectable amounts of nonself DNA are normally present on the skin surface of healthy adult individuals due to the adventitious transfer of DNA occurring during normal day‐to‐day social interactions, we designed an experiment in three phases. Phase 1 was used to deduce which DNA collection, extraction, and amplification methods were suited to investigating this question. During phase 2, the neck surface of 24 healthy adult volunteers was swabbed. DNA was extracted using the QIAamp DNA mini kit and amplified using the SGM Plus PCR amplification kit, using 28 PCR cycles. The work carried out during phase 3 involved a simulated assault to investigate primary and secondary transfer of DNA during physical contact. It was found that 23% of neck areas swabbed during phase 2 of this investigation showed nondonor alleles in the resulting DNA profile, with 5% of areas showing six or more nondonor alleles. The results of phase 3 showed that primary, secondary, and zero transfer of victim and/or offender DNA could be observed after physical contact and that alleles from an unknown source could still be detected in this more controlled experiment. The data presented in this paper demonstrate that DNA profiles generated after swabbing the skin surface of healthy adults can include components of an unknown source, present due to adventitious transfer. These components, if present in large quantities, have the potential to interfere with DNA profile interpretation of swabs taken for the investigation of physical assault by DNA profiling.
Estimating the Timing of Long Bone Fractures: Correlation Between the Postmortem Interval, Bone Moisture Content, and Blunt Force Trauma Fracture Characteristics*
Authors: Wieberg Danielle A.M. , Wescott Daniel J.
Published: July 2008

Abstract:  There is very limited knowledge about how long perimortem fracture characteristics persist into the postmortem interval (PMI). Therefore, in this study, 60 porcine long bones were exposed to natural taphonomic conditions and fractured with a steel bone breaking apparatus every 28 days throughout a 141‐day period. Differences between macroscopic blunt force trauma fracture characteristics (fracture angle, surface morphology, and outline) were examined to determine if they varied over time or in relationship to bone moisture content (ash weight) and overall assessment. There are significant relationships between (1) PMI and percent ash weight (%AW), fracture surface, and fracture angle and (2) %AW and fracture surface and fracture angle. Bone moisture content correlates significantly with fracture morphology and other characteristics commonly used by forensic anthropologists to determine the timing of traumatic injuries. However, fracture characteristics normally associated with perimortem trauma can persist long into the PMI.
Developing a method for laboratory biosafety testing impacts of transgenic plants on non‐target insects
Authors: Barratt B. I. P. , Crook K. E. , Ferguson C. M. , Johnstone P. D. , Malone L. A.
Published: July 2008

A series of laboratory experiments was carried out with the main objective of contributing to the development of biosafety testing methods for transgenic plants or plants produced by other novel technologies. The adult stage of a New Zealand native weevil, Nicaeana cervina (Broun) (Coleoptera: Curculionidae: Entiminae), was used as a test insect as this has become well‐adapted to introduced pasture species, feeding on both ryegrass [Lolium perenne L. (Poaceae)] and white clover [Trifolium repens L. (Fabaceae)]. Ryegrass plants used in experiments were infected with strains of the symbiotic endophytic fungi Neotyphodium lolii (Latch, M.J. Chr. & Samuels) and Epichloe festucae Leuchtm., Schardl & M.R. Siegel and included a transgenic endophyte, which was modified to eliminate production of the insect deterrent, peramine. Nicaeana cervina was exposed to these endophyte‐infected plants along with endophyte‐free ryegrass. Weevils were also exposed to white clover with high or low levels of cyanogens, and to white clover selections bred conventionally to contain high or low levels of crude protein. These bioassays were carried out sometimes over a series of time intervals, and weevil survival, feeding, and reproductive performance variables were measured. Nicaeana cervina survival was generally high, although feeding and oviposition were variable between experiments despite high replication in some cases. A summary of significant differences obtained across all experiments suggested that data on the proportion of weevils feeding and amounts of leaf material consumed were more often significantly different than treatment effects on survival or reproductive performance. A positive relationship between the proportion of weevils feeding and those ovipositing was found, suggesting that a 50% reduction in feeding would result in a 28% reduction in the proportion of weevils ovipositing. Power analysis using the range of values recorded for the variables measured indicated that for proportions of weevils surviving, feeding, or fecund, about 35–42 replicates would be required for a 40% difference in values. For measures of leaf consumption and numbers of eggs per female, fewer replicates were required for 95% power when these values were at the higher end of the range than when they were at the lower end of the observed range. The usefulness of the case study test insect and risk assessment protocols used for biosafety testing are discussed.
Povidone iodine causes opacification of silicone intraocular lens implants
Authors: Goel Siddharth , Kolli Lajipathi R. , Desai Shrivastav P. , Kumar Ashish , Gauba Vinod , Jayamanne Gerard R.
Published: July 2008

Purpose:  Four cases of silicone intraocular lens (IOL) opacifications presented at a routine cataract service at a UK district general hospital. A systematic investigation was performed to identify and eliminate the causative factor.

Methods:  An experiment was set up to determine the role of a chemically induced IOL injury. Silicone IOLs were exposed to the various chemical agents used during cataract surgery. One IOL was not exposed to any chemicals and was used as a control. The samples were then photographed with the same camera settings against the same background in a medical photography studio.

Results:  All samples photographed using this technique were clear as the unexposed control IOL, except the IOLs that had come into contact with povidone iodine (PI). Exposure to higher concentrations of PI appears to give a greater opacification and staining – a graded effect.

Conclusion:  While the toxic effects of PI on corneal endothelial health is well recognized, as far as the authors are aware no reports exists on the possible harmful effects of PI on IOLs. The results from the current study suggest that exposure of silicone IOLs to even small volumes of 5% PI can lead to IOL opacifying effects. Further studies are needed to determine the toxic effects of PI on all IOL materials. However, based on the results of our study, we strongly recommend extreme caution in the use of PI as prophylaxis against infection at the conclusion of cataract surgery and recommend great care to ensure complete wound closure.
Does exposure to dental education change the perceptions of Nigerian students to dentistry as a profession?
Authors: Sofola O. O. , Uti O. G. , Akpene O. I.
Published: July 2008

Aim:  To assess any effect of exposure to dental education and training on the perception and acceptance of dentistry by dental students at a Nigerian dental school.

Participants and methods:  The study was conducted by means of an anonymous self‐administered questionnaire distributed to second‐, fifth‐ and sixth‐year dental students at the College of Medicine, University of Lagos. Participation was voluntary and 67 students agreed to participate in the study. Majority (51%) were aged between 24 and 27 years. The questionnaire contained questions on demography, preferred choice of course on admission, attempts at change of course, their present perception to dentistry and future dental practice.

Result:  Over 90% of them were admitted through the Joint admission and matriculation board and 40 (60%) chose Medicine as their first course choice. Of the 40, 18 had attempted a change of course and eight were still interested in a career change (four were in the fifth year). Most of the change in attitude occurred in fourth year. Sixty (90%) intend to practice dentistry but 38 (63%) of these would like to practice outside Nigeria.

Conclusion:  Exposure to dental training appears to improve students’ perception about dental practice but uncertainty about future prospects in Nigeria may lead to brain drain in the dental profession.
Injectable growth/differentiation factor‐5–recombinant human collagen composite induces endochondral ossification via Sry‐related HMG box 9 (Sox9)expression and angiogenesis in murine calvariae
Authors: Kadomatsu H. , Matsuyama T. , Yoshimoto T. , Negishi Y. , Sekiya H. , Yamamoto M. , Izumi Y.
Published: July 2008

Background and Objective:  The types of collagens available today as biomaterials are purified from animal tissues. A major growing concern, however, is their safety, since there are risks of viral and prion contamination and of unknown and potentially zoonotic infectious diseases. The present study aimed to assess, using immunohistochemistry, the effects of recombinant human growth/differentiation factor‐5 (rhGDF‐5) combined with recombinant human collagen I (rhCI) on bone formation in murine calvariae.

Material and Methods:  Composite rhGDF‐5–rhCI or rhCI alone was injected subcutaneously into murine calvariae. After 3, 7 or 14 days, tissues were examined radiologically, histologically and immunohistochemically. The production of vascular endothelial growth factor (VEGF) by primary osteoblasts, periosteal cells and connective tissue fibroblasts isolated enzymatically from neonatal murine calvariae was also assessed.

Results:  A protrusion was observed on the calvariae at the site injected with rhGDF‐5/rhCI composite. Its mineral density was shown to be different from that of the existing bone by two‐dimensional microcomputed tomography. Type II collagen‐positive staining was restricted to newly formed tissues. Thus, the newly formed tissues seemed to be bone‐ and cartilage‐like tissues. A number of vessels with positively stained cells for Von Willebrand factor were detected in the newly formed tissues. The rhGDF‐5 enhanced VEGF production in cultured connective tissue fibroblasts. Sry‐related HMG box 9 (Sox9)‐positive cells were detected in the hypertrophic periosteum, and penetrated into the newly formed tissues.

Conclusions:  These results suggest that rhCI seems to allow the release of rhGDF‐5 and that rhGDF‐5–rhCI composite induces endochondral ossification via Sox9 expression and angiogenesis in murine calvariae.
Areca nut extract represses migration and differentiation while activating matrix metalloproteinase‐9 of normal gingival epithelial cells
Authors: Tseng Y‐H , Chang K‐W , Liu C‐J , Lin C‐Y , Yang S‐C , Lin S‐C
Published: July 2008

Background and Objective:  Areca (betel) chewing is associated with an increase in the incidence of periodontal diseases. Aberrations in matrix metalloproteinase (MMP) expression have been reported to be associated with periodontal disease. This study investigated the effects of areca nut extract on MMP activity and the phenotype of human gingival epithelial cells.

Material and Methods:  Reverse transcription‐polymerase chain reaction, western blotting and gelatin zymography were used to assay MMPs. Cell viability, mobility and detachment assays were performed to characterize the phenotypic impact. Confocal microscopy was employed to evaluate cell aggregation and the distribution of E‐cadherin and F‐actin.

Results:  Treatment of gingival epithelial cells with 10 µg/mL of areca nut extract reduced its cell viability. Treatment with 5 and 10 µg/mL of areca nut extract for 24 h activated MMP‐9 but not MMP‐2 in gingival epithelial cells. This activation could be nuclear factor‐κB dependent and was abrogated by 10 µm curcumin. Areca nut extract also reduced the migration and detachment of gingival epithelial cells. The differentiated cell–cell contact of gingival epithelial cells was markedly impaired by areca nut extract. This was accompanied by a disruption of distribution of E‐cadherin and F‐actin.

Conclusion:  The areca nut extract‐mediated activation of MMP‐9 in gingival epithelial cells could signify a potential periodontal pathogenesis in areca chewers. The areca nut extract‐mediated inhibition of cell viability and migration, together with the changed aggregation in gingival epithelial cells, suggests that impairment of the re‐epithelization underlies the process and this, in turn, might exacerbate gingival inflammation.
Tetracycline release from tripolyphosphate–chitosan cross‐linked sponge: a preliminary in vitro study
Authors: Shen E‐C. , Wang C. , Fu E. , Chiang C‐Y. , Chen T‐T. , Nieh S.
Published: July 2008

Background and Objective:  The aim of this study was to design a tripolyphosphate–chitosan cross‐linked tetracycline‐containing (TPP‐TC) sponge that slowly releases tetracycline, for future periodontal applications.

Material and methods:  Chitosan sponge was made by freezing and drying 2.5% chitosan solution. Tripolyphosphate‐chitosan cross‐linked (TPP) sponge was made by immersing the chitosan sponge in tripolyphosphate solution and air drying it. Tetracycline‐containing chitosan (TC) sponge was prepared by freezing and drying a mixture of chitosan and tetracycline. TPP‐TC sponge was made by immersing the TC sponge in tripolyphosphate solution. The weight, thickness and diameter of the four chitosan sponges were recorded. Their surface microstructures were inspected using scanning electron microscopy. The amount of tetracycline released from the sponges was analyzed by spectrophotometry. Antimicrobial activities of the residual sponges were tested against Staphylococcus aureus and Escherichia coli.

Results:  The topography of the scaffolds was intact after the addition of tetracycline. However, increased surface irregularities were noted. In sponges with tripolyphosphate, intensified surface folding was observed. The weight of the sponges increased after tripolyphosphate and tetracycline were added, but their thicknesses and diameters decreased after cross‐linking. Tetracycline was detected in the solution containing TPP‐TC sponges until day 11. On day 7, the tetracycline released from TC sponges was less than that released from TPP‐TC sponges. Bacterial growth was inhibited by sponges containing tetracycline. The inhibitory effect of the TPP‐TC sponges was detectable until day 11.

Conclusion:  Our data showed that TPP‐TC sponge was suitable as a slow‐release device for tetracycline and that it maintained antimicrobial effects against the bacteria tested for up to 11 d.
Fractal analysis of dental radiographs to detect periodontitis‐induced trabecular changes
Authors: Updike Sophia Xiang , Nowzari Hessam
Published: July 2008

Background and Objective:  The aim was to evaluate fractal analysis as a tool to quantitatively measure the impact of periodontal disease on surrounding bone. The diagnosis of periodontitis is based on information obtained from clinical and radiographic examinations. The current standard use of dental radiographs is visual inspection, often with no quantitative analysis. Fractal analysis can be used to examine trabecular bone patterns among periodontal patients.

Material and Methods:  Patients (n = 108) from the University of Southern California School of Dentistry were classified into three groups: healthy, moderate and severe periodontitis. A region of interest was selected from periapical radiographs. Image processing was applied to correct for lighting irregularity, and the box‐counting method was used to calculate a fractal dimension. ANOVA and ANCOVA were used to measure fractal dimension differences between all groups.

Results:  According to the statistical tests, significant differences in average fractal dimensions were measured between healthy and moderate periodontitis groups (p < 0.01) and between healthy and severe periodontitis groups (p < 0.001). Higher fractal dimensions were measured in healthy periodontal patients.

Conclusion:  Fractal analysis evidenced significant differences between patients affected and not affected by periodontitis. The box‐counting method quantitatively describes the severity of bone disease and can be used to improve current diagnostic techniques.
Putative stem cells in regenerating human periodontium
Authors: Lin N‐H. , Menicanin D. , Mrozik K. , Gronthos S. , Bartold P. M.
Published: July 2008

Background and Objective:  Human postnatal stem cells have been identified in periodontal ligament, with the potential to regenerate the periodontium in vivo. However, it is unclear if periodontal ligament stem cells are present in regenerating periodontal tissues. The aim of this study was to identify and localize putative stem cells in block biopsies and explant cultures of regenerating human periodontal tissues.

Material and Methods:  Guided tissue regeneration was carried out on the molars of three human volunteers. After 6 wk, the teeth with the surrounding regenerating tissues and bone were surgically removed and processed for immunohistochemistry. The mesenchymal stem cell‐associated markers STRO‐1, CD146 and CD44 were used to identify putative stem cells. Cell cultures established from regenerating tissue explants were analysed by flow cytometry to assess the expression of these markers. Mineralization, calcium concentration and adipogenic potential of regenerating tissue cells were assessed and compared with periodontal ligament stem cells, bone marrow stromal stem cells and gingival fibroblasts.

Results:  STRO‐1+, CD44+ and CD146+ cells were identified in the regenerating tissues. They were found mainly in the paravascular and extravascular regions. Flow cytometry revealed that cultured regenerating tissue cells expressed all three mesenchymal stem cell associated markers. The regenerating tissue cells were able to form mineral deposits and lipid‐containing adipocytes. However, the level of mineralization in these cells was lower than that of periodontal ligament stem cells and bone marrow stromal stem cells.

Conclusion:  Cells with characteristics of putative mesenchymal stem cells were found in regenerating periodontal tissues, implying their involvement in periodontal regeneration.
Human periodontal ligament: a niche of neural crest stem cells
Authors: Coura G. S. , Garcez R. C. , De Aguiar C. B. N. Mendes , Alvarez‐Silva M. , Magini R. S. , Trentin A. G.
Published: July 2008

Background and Objective:  The periodontal ligament is a specialized connective tissue, derived from dental follicle and originated from neural crest cells. Recently it has been suggested, based on animal models, that periodontal ligament could be a niche for neural crest stem cells. However, there is still little knowledge on this subject. The identification of neural crest adult stem cells has received much attention based on its potential in tissue regeneration. The objective of the present work was to verify the human periodontal ligament as a niche for neural crest stem cells.

Material and Methods:  Cells from human periodontal ligament were isolated from 10 teeth of seven individuals (periodontal ligament pool group) and also from four teeth of one individual (periodontal ligament single group), after enzymatic digestion. The cells were cultured in specific inductive medium. Analyses of protein and gene expression were performed through immunocytochemistry and reverse transcription–polymerase chain reaction techniques, respectively.

Results:  Mesodermal phenotypes (adipogeneic, osteogenic and myofibroblastic) were identified after culture in inductive medium. Immunocytochemistry analyses showed the presence of the nestin marker of neural stem cells and also markers of undifferentiated neural crest cells (HNK1, p75). When cultured in inductive medium that allowed neural differentiation, the cells showed markers for β‐tubulin III, neurofilament M, peripherin, microtubule‐associated protein 2 and protein zero. The results were similar between the two study groups (the periodontal ligament pool group and the periodontal ligament single group).

Conclusion:  This research provides evidence that human periodontal ligament, in addition to its mesodermal derivatives, produces neural crest‐like cells. Such features suggest a recapitulation of their embryonic state. The human periodontal ligament revealed itself as a viable alternative source for possible primitive precursors to be used in stem‐cell therapies.
Insulin‐like growth factor binding protein‐5 enhances the migration and differentiation of gingival epithelial cells
Authors: Hung P.‐S. , Kao S.‐Y. , Liu C.‐J. , Tu H.‐F. , Wu C.‐H. , Lin S.‐C.
Published: July 2008

Background and Objective:  The objective was to define the roles of insulin‐like growth factor binding protein‐5 (IGFBP‐5) in gingival epithelial cells (GEC). Human IGFBP‐5 is expressed in many cell types and has diverse biological functions. It stimulates the growth of bone cells and is associated with the impedance of gingival fibroblast apoptosis. In gingival epithelium, IGFBP‐5 is expressed in the cells of the differentiated stratum spinosum layer.

Material and Methods:  Recombinant IGFBP‐5 protein treatment and knockdown of IGFBP‐5 expression using a lentivirus‐delivered short hairpin RNA was carried out in human GEC. Proliferation, apoptosis, anoikis, migration, differentiation and gene expression in GEC were analyzed and molecular images were obtained.

Results:  The IGFBP‐5 had no effect on proliferation, but it slightly suppressed apoptosis and anoikis of GEC. It also induced GEC migration and upregulated the expression of involucrin, transglutaminase‐1, keratin and focal adhesion kinase. The IGFBP‐5 induced migration partly via an insulin‐like growth factor‐independent mechanism. The knockdown of IGFBP‐5 downregulated the expression of involucrin, transglutaminase‐1 and focal adhesion kinase.

Conclusion:  Expression of IGFBP‐5 in GEC is associated with anti‐apoptosis, migration and differentiation of GEC. These phenotypic effects may be associated with focal adhesion kinase and are advantageous for re‐epithelization of GEC and the maintenance of gingival health.
Periodontal tissue engineering by transplantation of multilayered sheets of phenotypically modified gingival fibroblasts
Authors: Nakajima K. , Abe T. , Tanaka M. , Hara Y.
Published: July 2008

Background and Objective:  In periodontal tissue engineering, the sourcing of most of the relevant cells is limited by poor accessibility, whereas the use of readily available gingival fibroblasts is hampered because of their inhibitory effects on bone formation. To address the latter drawback, we developed a new graft composed of fibronectin (FN) matrix‐based multilayered cell sheets of human gingival fibroblasts modified to express alkaline phosphatase (ALP). This study was undertaken to investigate the effects of this graft, called the FN‐ALP transplant, on the healing of periodontal defects in a rat model.

Material and Methods:  The FN‐ALP transplants were grafted into periodontal fenestration bone defects in immunosuppressed rats. The process of periodontal healing was examined by histology, histomorphometry and immunohistochemistry. Grafted cells were tracked by immunostaining with human‐specific antibodies. Control groups included non‐transplanted empty defects and defects to which cell sheets without ALP induction had been grafted.

Results:  After implantation, the FN‐ALP transplants healed alveolar bone defects by intramembranous ossification, with formation of cementum and periodontal ligament. Moreover, FN‐ALP transplants increased new bone formation, by endochondral ossification, on the mandibular cortex adjacent to the defect. Grafted fibroblasts were located near host osteoblasts and chondrocyte precursor cells early in the ossification process but were undetectable on and in newly formed bone and cartilage.

Conclusion:  These results indicate that the FN‐ALP transplants support alveolar bone regeneration within the defect and augment bone formation outside the defect through the recruitment of host osteo/chondrogenic cells, suggesting their potential for periodontal tissue engineering applications.
Variability in the response of human dendritic cells stimulated with Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans
Authors: Vernal R. , Leon R. , Herrera D. , Garcia‐Sanz J. A. , Silva A. , Sanz M.
Published: July 2008

Background and Objective:  Dendritic cells are able to prime and polarize naïve T cells towards either a T helper 1 or a T helper 2 response, depending on the antigen type and concentration, the costimulatory signals and the local cytokine millieu. In this investigation, we analyzed the response of human dendritic cells to stimulation with different concentrations of Porphyromonas gingivalis or Aggregatibacter actinomycetemcomitans.

Material and Methods:  Using different concentrations of P. gingivalis ATCC 33277 and A. actinomycetemcomitans ATCC 33384, we determined the expression of the maturation markers CD80 and CD86 from purified human dendritic cells by flow cytometry. We also evaluated the mRNA expression levels for the cytokines interleukin‐1β, interleukin‐2, interleukin‐5, interleukin‐6, interleukin‐10, interleukin‐12, interleukin‐13, interferon‐γ, tumor necrosis factor‐α and tumor necrosis factor‐β by quantitative real‐time reverse transcription–polymerase chain reaction.

Results:  Both P. gingivalis and A. actinomycetemcomitans led to dendritic cell maturation, but the expression of CD80 was higher when the dendritic cells were stimulated with A. actinomycetemcomitans. Although both pathogens induced a T helper 1 pattern of cytokine expression, A. actinomycetemcomitans‐stimulated dendritic cells expressed interleukin‐1β, interleukin‐12, interferon‐γ, tumor necrosis factor‐α and tumor necrosis factor‐β at lower bacterial concentrations than P. gingivalis. While 106 bacteria/mL of P. gingivalis or 104 bacteria/mL of A. actinomycetemcomitans induced expression of interleukin‐12p40, the expression of other cytokines required 10 to 100‐fold higher concentrations of bacteria.

Conclusion:  These results demonstrate that A. actinomycetemcomitans is a more potent immunogen than P. gingivalis because, at least in vitro, it induces stronger differentiation and activation of dendritic cells. In addition, our data also show that for a given strain, the bacterial load determines the pattern of cytokines that are expressed.
A novel selective medium for isolation of Aggregatibacter (Actinobacillus) actinomycetemcomitans
Authors: Tsuzukibashi O , Takada K , Saito M , Kimura C , Yoshikawa T , Makimura M , Hirasawa M
Published: July 2008

Background and Objective:  Conventional selective media have been used for the selection of Aggregatibacter (Actinobacillus) actinomycetemcomitans in clinical samples. The proportion of A. actinomycetemcomitans grown on the selective media in vitro may not reflect the true counts in vivo because of the low selectivity. A novel selective medium, designated AASM, was developed for the isolation of A. actinomycetemcomitans.

Material and Methods:  AASM was prepared by adding of 200 μg/mL of vancomycin and 10 U/mL of bacitracin to AAGM, which contains dextrose, sodium bicarbonate, trypticase soy, yeast extract and agar. Clinical efficacy was evaluated by the recovery, on AASM, of A. actinomycetemcomitans from subgingival samples of 44 periodontally healthy subjects and 76 patients with chronic periodontitis.

Results:  All serotypes (a–f) of A. actinomycetemcomitans strains grew well, and the average growth recovery of A. actinomycetemcomitans on AASM medium was 94.4% (80.0–109.7%) of that on AAGM. The exclusive rate of other bacteria was 99.9% in clinical samples cultured on AASM. A. actinomycetemcomitans was not detected in periodontally healthy persons but was detected in 25 (32.9%) patients with chronic periodontitis. The predominant serotype was c, detected in 11 subjects.

Conclusion:  The new selective medium, AASM, was highly selective for A. actinomycetemcomitans, eliminated possible false‐positive results and was useful for the isolation of A. actinomycetemcomitans from clinical samples.
Analysis of the capsular polysaccharide biosynthesis locus of Porphyromonas gingivalis and development of a K1‐specific polymerase chain reaction‐based serotyping assay
Authors: Brunner J. , Crielaard W. , Van Winkelhoff A. J.
Published: July 2008

Background and Objective:  Porphyromonas gingivalis is a gram‐negative obligate anaerobe that is strongly associated with severe periodontitis. Previous reports showed an association of P.  gingivalis capsular polysaccharide with virulence. The K1 capsular polysaccharide was found to be more immunostimulatory than the other serotypes. Our objective was to explore the genetic background of the capsule biosynthesis (K‐antigen) locus in a representative group of K1 serotype strains.

Material and Methods:  We used restriction fragment length polymorphism, polymerase chain reaction (PCR) and DNA sequencing to study the capsular polysaccharide locus in P. gingivalis K1 strains. For serotyping by double immunodiffusion and PCR we used 32 strains of P. gingivalis, including strains of all six known K serotypes.

Results:  All tested K1 strains showed high conservation of the capsular polysaccharide locus, although a DNA re‐arrangement was found in two strains. Based on this information a K1‐specific PCR‐based serotyping assay was designed. The specificity and sensitivity of this test were confirmed using non‐K1 P. gingivalis serotypes.

Conclusion:  The capsular polysaccharide locus of P. gingivalis is conserved but may vary slightly among K1 strains. The new K1 serotyping assay presented here is much faster than double immunodiffusion and can detect K1 strains in a very selective and sensitive way. This method may therefore be clinically relevant in the detection of the virulent P. gingivalis K1 serotype.
Interleukin‐4, a T‐helper 2 cell cytokine, is associated with the remission of periodontal disease
Authors: Pradeep A. R. , Roopa Y. , Swati P. P.
Published: July 2008

Background and Objectives:  Interleukin‐4 (IL‐4), secreted mainly by T‐helper 2 cells, is a key cytokine for the growth and proliferation of B lymphocytes. Previous studies have proved that IL‐4 has an anti‐inflammatory effect owing to its efficient inhibition of the production of proinflammatory cytokines such as tumour necrosis factor‐α (TNF‐α), IL‐1α, IL‐1β, IL‐6 and IL‐8 by monocytes/macrophages. The aim of the present study was to assess the relation between clinical parameters and concentrations of IL‐4 within gingival crevicular fluid from inflamed gingiva and periodontitis sites and, subsequently, after treatment of the periodontitis sites.

Material and Methods:  A total of 60 subjects were divided into three groups based on gingival index (GI), pocket probing depth and clinical attachment loss (CAL): healthy (group 1), gingivitis (group 2) and chronic periodontitis (group 3). A fourth group (group 4) consisted of 20 subjects from group 3, 6–8 weeks after treatment (i.e. scaling and root planing). Gingival crevicular fluid samples collected from each patient were quantified for IL‐4 using the enzymatic immunometric assay.

Results:  The highest mean concentration of IL‐4 was obtained for group 1 (99.39 ± 49.33 pg/mL) and the lowest mean concentration of IL‐4 was obtained for group 3 (15.78 ± 21.92 pg/mL). The mean IL‐4 concentrations for group 2 (64.34 ± 39.56 pg/mL) and group 4 (68.92 ± 42.85 pg/mL) were intermediate between the levels in healthy subjects and periodontitis subjects.

Conclusion:  The mean concentration of IL‐4 decreased from periodontal health to disease. Thus, we suggest that type 2 helper T cell cytokine, as represented by IL‐4, was associated with the remission or improvement of periodontal disease.
CXCL13 expression and follicular dendritic cells in relation to B‐cell infiltration in periodontal disease tissues
Authors: Nakajima T. , Amanuma R. , Ueki‐Maruyama K. , Oda T. , Honda T. , Ito H. , Yamazaki K.
Published: July 2008

Background and Objective:  B lymphocyte is the dominant infiltrating cell type in periodontitis lesions. CXCL13, produced by follicular dendritic cells, endothelial cells and fibroblasts, is crucial for B‐cell trafficking. An association between chronic inflammation and lymphoid organogenesis has been reported in infection and in autoimmune responses, in which T‐cell/B‐cell follicles with a follicular dendritic cell network are formed. The aim of this study was to examine CXCL13 expression and follicular dendritic cell distribution in relation to B‐cell infiltration in chronic inflammatory periodontal lesions.

Material and Methods:  Fifty‐eight gingival tissue biopsies from patients with periodontitis and 25 samples from subjects with gingivitis were analyzed. Gene expression for CXCL13 and for the CD21 long isoform was analyzed using the reverse transcription–polymerase chain reaction. Immunohistochemical analysis was performed using antibodies to CXCL13, CXCR5, follicular dendritic cells, CD3 and CD19 on serial cryostat sections.

Results:  mRNA for CXCL13 was expressed in both periodontitis and gingivitis tissues. The number of CXCL13+ cells was significantly higher in periodontitis than in gingivitis in connective tissues subjacent to the pocket epithelium and positively correlated with the number of CD19+ cells. CXCL13+ cells were distributed in B‐cell‐dominant areas both with and without follicular dendritic cells. Although obvious reticular networks of follicular dendritic cells were not found in periodontitis and gingivitis, the accumulation of follicular dendritic cells in B‐cell‐dominant areas in periodontitis was observed in some patients.

Conclusion:  These findings suggested that CXCL13 and follicular dendritic cells were involved in B‐cell recruitment to, and B‐cell distribution in, chronic inflammatory periodontal lesions.