In vitro characterization of a synthetic calcium phosphate bone graft on periodontal ligament cell and osteoblast behavior and its combination with an enamel matrix derivative.

OBJECTIVES Recent studies suggest that a combination of enamel matrix derivative (EMD) with grafting material may improve periodontal wound healing/regeneration. Newly developed calcium phosphate (CaP) ceramics have been demonstrated a viable synthetic replacement option for bone grafting filler materials. AIMS This study aims to test the ability for EMD to adsorb to the surface of CaP particles and to determine the effect of EMD on downstream cellular pathways such as adhesion, proliferation, and differentiation of primary human osteoblasts and periodontal ligament (PDL) cells. MATERIALS AND METHODS EMD was adsorbed onto CaP particles and analyzed for protein adsorption patterns via scanning electron microscopy and high-resolution immunocytochemistry with an anti-EMD antibody. Cell attachment and cell proliferation were quantified using CellTiter 96 One Solution Cell Assay (MTS). Cell differentiation was analyzed using real-time PCR for genes encoding Runx2, alkaline phosphatase, osteocalcin, and collagen1α1, and mineralization was assessed using alizarin red staining. RESULTS Analysis of cell attachment revealed significantly higher number of cells attached to EMD-adsorbed CaP particles when compared to control and blood-adsorbed samples. EMD also significantly increased cell proliferation at 3 and 5 days post-seeding. Moreover, there were significantly higher mRNA levels of osteoblast differentiation markers including collagen1α1, alkaline phosphatase, and osteocalcin in osteoblasts and PDL cells cultured on EMD-adsorbed CaP particles at various time points. CONCLUSION The present study suggests that the addition of EMD to CaP grafting particles may influence periodontal regeneration by stimulating PDL cell and osteoblast attachment, proliferation, and differentiation. Future in vivo and clinical studies are required to confirm these findings. CLINICAL RELEVANCE The combination of EMD and CaP may represent an option for regenerative periodontal therapy in advanced intrabony defects.

Beneficial effects of adiponectin on periodontal ligament cells under normal and regenerative conditions

Type 2 diabetes and obesity are increasing worldwide and linked to periodontitis, a chronic disease which is characterized by the irreversible destruction of the tooth-supporting tissues, that is, periodontium. The mechanisms underlying the association of diabetes mellitus and obesity with periodontal destruction and compromised periodontal healing are not well understood, but decreased plasma levels of adiponectin, as found in diabetic and obese individuals, might be a critical mechanistic link. The aim of this in vitro study was to examine the effects of adiponectin on periodontal ligament (PDL) cells under normal and regenerative conditions, and to study the regulation of adiponectin and its receptors in these cells. Adiponectin stimulated significantly the expression of growth factors and extracellular matrix, proliferation, and in vitro wound healing, reduced significantly the constitutive tumor necrosis factor-α expression, and caused a significant upregulation of its own expression. The beneficial actions of enamel matrix derivative on a number of PDL cell functions critical for periodontal regeneration were partially enhanced by adiponectin. The periodontopathogen Porphyromonas gingivalis inhibited the adiponectin expression and stimulated the expression of its receptors. In conclusion, reduced levels of adiponectin, as found in type 2 diabetes and obesity, may compromise periodontal health and healing.

In vitro-activity of oily calcium hydroxide suspension on microorganisms as well as on human alveolar osteoblasts and periodontal ligament fibroblasts.

BACKGROUND Findings from animal and human studies have indicated that an oily calcium hydroxide suspension (OCHS) may improve early wound healing in the treatment of periodontitis. Calcium hydroxide as the main component is well known for its antimicrobial activity, however at present the effect of OCHS on the influence of periodontal wound healing/regeneration is still very limited. The purpose of this in vitro study was to investigate the effect of OCHS on periodontopathogenic bacteria as well as on the attachment and proliferation of osteoblasts and periodontal ligament fibroblasts. METHODS Human alveolar osteoblasts (HAO) and periodontal ligament (PDL) fibroblasts were cultured on 3 concentrations of OCHS (2.5, 5 and 7.5 mg). Adhesion and proliferation were counted up to 48 h and mineralization was assayed after 1 and 2 weeks. Furthermore potential growth inhibitory activity on microorganisms associated with periodontal disease (e.g. Porphyromonas gingivalis, Tannerella forsythia, Aggregatibacter actinomycetemcomitans) as well as the influence of periodontopathogens and OCHS on the HAO and PDL fibroblasts counts were determined. RESULTS More than a 2-fold increase in adherent HAO cells was observed at 4 h following application of OCHS when compared to the control group (p = 0.007 for 2.5 mg). Proliferation of HAO cells at 48 h was stimulated by moderate concentrations (2.5 mg; 5 mg) of OCHS (each p < 0.001), whereas a high concentration (7.5 mg) of OCHS was inhibitory (p = 0.009). Mineralization was observed only for HAO cells treated with OCHS. OCHS did not exert any positive effect on attachment or proliferation of PDL fibroblasts. Although OCHS did not have an antibacterial effect, it did positively influence attachment and proliferation of HAO cells and PDL fibroblasts in the presence of periodontopathogens. CONCLUSIONS The present data suggests that OCHS promotes osteoblast attachment, proliferation and mineralization in a concentration-dependent manner and results are maintained in the presence of periodontal pathogens.

Proteome-wide screening of the European porcine reproductive and respiratory syndrome virus reveals a broad range of T cell antigen reactivity.

The porcine reproductive and respiratory syndrome virus (PRRSV) is a rapidly evolving and diversifying pathogen necessitating the development of improved vaccines. Immunity to PRRSV is not well understood although there are data suggesting that virus-specific T cell IFN-γ responses play an important role. We therefore aimed to better characterise the T cell response to genotype 1 (European) PRRSV by utilising a synthetic peptide library spanning the entire proteome and a small cohort of pigs rendered immune to PRRSV-1 Olot/91 by repeated experimental infection. Using an IFN-γ ELISpot assay as a read-out, we were able to identify 9 antigenic regions on 5 of the viral proteins and determine the corresponding responder T cell phenotype. The diversity of the IFN-γ response to PRRSV proteins suggests that antigenic regions are scattered throughout the proteome and no one single antigen dominates the T cell response. To address the identification of well-conserved T cell antigens, we subsequently screened groups of pigs infected with a closely related avirulent PRRSV-1 strain (Lelystad) and a divergent virulent subtype 3 strain (SU1-Bel). Whilst T cell responses from both groups were observed against many of the antigens identified in the first study, animals infected with the SU1-Bel strain showed the greatest response against peptides representing the non-structural protein 5. The proteome-wide peptide library screening method used here, as well as the antigens identified, warrant further evaluation in the context of next generation vaccine development.

Virulence of broad- and narrow-host-range Salmonella enterica serovars in the streptomycin-pretreated mouse model.

Salmonella enterica subspecies I serovars are common bacterial pathogens causing diseases ranging from enterocolitis to systemic infections. Some serovars are adapted to specific hosts, whereas others have a broad host range. The molecular mechanisms defining the virulence characteristics and the host range of a given S. enterica serovar are unknown. Streptomycin pretreated mice provide a surrogate host model for studying molecular aspects of the intestinal inflammation (colitis) caused by serovar Typhimurium (S. Hapfelmeier and W. D. Hardt, Trends Microbiol. 13:497-503, 2005). Here, we studied whether this animal model is also useful for studying other S. enterica subspecies I serovars. All three tested strains of the broad-host-range serovar Enteritidis (125109, 5496/98, and 832/99) caused pronounced colitis and systemic infection in streptomycin pretreated mice. Different levels of virulence were observed among three tested strains of the host-adapted serovar Dublin (SARB13, SD2229, and SD3246). Several strains of host restricted serovars were also studied. Two serovar Pullorum strains (X3543 and 449/87) caused intermediate levels of colitis. No intestinal inflammation was observed upon infection with three different serovar Paratyphi A strains (SARB42, 2804/96, and 5314/98) and one serovar Gallinarum strain (X3796). A second serovar Gallinarum strain (287/91) was highly virulent and caused severe colitis. This strain awaits future analysis. In conclusion, the streptomycin pretreated mouse model can provide an additional tool to study virulence factors (i.e., those involved in enteropathogenesis) of various S. enterica subspecies I serovars. Five of these strains (125109, 2229, 287/91, 449/87, and SARB42) are subject of Salmonella genome sequencing projects. The streptomycin pretreated mouse model may be useful for testing hypotheses derived from this genomic data.

A Biofilm Pocket Model to Evaluate Different Non-Surgical Periodontal Treatment Modalities in Terms of Biofilm Removal and Reformation, Surface Alterations and Attachment of Periodontal Ligament Fibroblasts.

BACKGROUND AND AIM There is a lack of suitable in vitro models to evaluate various treatment modalities intending to remove subgingival bacterial biofilm. Consequently, the aims of this in vitro-study were: a) to establish a pocket model enabling mechanical removal of biofilm and b) to evaluate repeated non-surgical periodontal treatment with respect to biofilm removal and reformation, surface alterations, tooth hard-substance-loss, and attachment of periodontal ligament (PDL) fibroblasts. MATERIAL AND METHODS Standardized human dentin specimens were colonized by multi-species biofilms for 3.5 days and subsequently placed into artificially created pockets. Non-surgical periodontal treatment was performed as follows: a) hand-instrumentation with curettes (CUR), b) ultrasonication (US), c) subgingival air-polishing using erythritol (EAP) and d) subgingival air-polishing using erythritol combined with chlorhexidine digluconate (EAP-CHX). The reduction and recolonization of bacterial counts, surface roughness (Ra and Rz), the caused tooth substance-loss (thickness) as well as the attachment of PDL fibroblasts were evaluated and statistically analyzed by means of ANOVA with Post-Hoc LSD. RESULTS After 5 treatments, bacterial reduction in biofilms was highest when applying EAP-CHX (4 log10). The lowest reduction was found after CUR (2 log10). Additionally, substance-loss was the highest when using CUR (128±40 µm) in comparison with US (14±12 µm), EAP (6±7 µm) and EAP-CHX (11±10) µm). Surface was roughened when using CUR and US. Surfaces exposed to US and to EAP attracted the highest numbers of PDL fibroblasts. CONCLUSION The established biofilm model simulating a periodontal pocket combined with interchangeable placements of test specimens with multi-species biofilms enables the evaluation of different non-surgical treatment modalities on biofilm removal and surface alterations. Compared to hand instrumentation the application of ultrasonication and of air-polishing with erythritol prevents from substance-loss and results in a smooth surface with nearly no residual biofilm that promotes the reattachment of PDL fibroblasts.

The broad-leaf herbicide 2,4-dichlorophenoxyacetic acid turns rice into a living trap for a major insect pest and a parasitic wasp

Synthetic chemical elicitors of plant defense have been touted as a powerful means for sustainable crop protection. Yet, they have never been successfully applied to control insect pests in the field. We developed a high-throughput chemical genetics screening system based on a herbivore-induced linalool synthase promoter fused to a β-glucuronidase (GUS) reporter construct to test synthetic compounds for their potential to induce rice defenses. We identified 2,4-dichlorophenoxyacetic acid (2,4-D), an auxin homolog and widely used herbicide in monocotyledonous crops, as a potent elicitor of rice defenses. Low doses of 2,4-D induced a strong defensive reaction upstream of the jasmonic acid and ethylene pathways, resulting in a marked increase in trypsin proteinase inhibitor activity and volatile production. Induced plants were more resistant to the striped stem borer Chilo suppressalis, but became highly attractive to the brown planthopper Nilaparvata lugens and its main egg parasitoid Anagrus nilaparvatae. In a field experiment, 2,4-D application turned rice plants into living traps for N. lugens by attracting parasitoids. • Our findings demonstrate the potential of auxin homologs as defensive signals and show the potential of the herbicide to turn rice into a selective catch crop for an economically important pest.

A Lentiviral Vector Expressing Japanese Encephalitis Virus-like Particles Elicits Broad Neutralizing Antibody Response in Pigs.

BACKGROUND Japanese encephalitis virus (JEV) is the major cause of viral encephalitis in Southeast Asia. Vaccination of domestic pigs has been suggested as a "one health" strategy to reduce viral disease transmission to humans. The efficiency of two lentiviral TRIP/JEV vectors expressing the JEV envelope prM and E glycoproteins at eliciting protective humoral response was assessed in a mouse model and piglets. METHODOLOGY/PRINCIPAL FINDINGS A gene encoding the envelope proteins prM and E from a genotype 3 JEV strain was inserted into a lentiviral TRIP vector. Two lentiviral vectors TRIP/JEV were generated, each expressing the prM signal peptide followed by the prM protein and the E glycoprotein, the latter being expressed either in its native form or lacking its two C-terminal transmembrane domains. In vitro transduction of cells with the TRIP/JEV vector expressing the native prM and E resulted in the efficient secretion of virus-like particles of Japanese encephalitis virus. Immunization of BALB/c mice with TRIP/JEV vectors resulted in the production of IgGs against Japanese encephalitis virus, and the injection of a second dose one month after the prime injection greatly boosted antibody titers. The TRIP/JEV vectors elicited neutralizing antibodies against JEV strains belonging to genotypes 1, 3, and 5. Immunization of piglets with two doses of the lentiviral vector expressing JEV virus-like particles led to high titers of anti-JEV antibodies, that had efficient neutralizing activity regardless of the JEV genotype tested. CONCLUSIONS/SIGNIFICANCE Immunization of pigs with the lentiviral vector expressing JEV virus-like particles is particularly efficient to prime antigen-specific humoral immunity and trigger neutralizing antibody responses against JEV genotypes 1, 3, and 5. The titers of neutralizing antibodies elicited by the TRIP/JEV vector are sufficient to confer protection in domestic pigs against different genotypes of JEV and this could be of a great utility in endemic regions where more than one genotype is circulating.

Effect of Enamel Matrix Derivative Liquid on Osteoblast and Periodontal Ligament Cell Proliferation and Differentiation.

BACKGROUND Enamel matrix derivatives (EMDs) have been used clinically for more than a decade for the regeneration of periodontal tissues. The aim of the present study is to analyze the effect on cell growth of EMDs in a gel carrier in comparison to EMDs in a liquid carrier. EMDs in a liquid carrier have been shown to adsorb better to bone graft materials. METHODS Primary human osteoblasts and periodontal ligament (PDL) cells were exposed to EMDs in both gel and liquid carriers and compared for their ability to induce cell proliferation and differentiation. Alizarin red staining and real-time polymerase chain reaction for expression of genes encoding collagen 1, osteocalcin, and runt-related transcription factor 2, as well as bone morphogenetic protein 2 (BMP2), transforming growth factor (TGF)-β1, and interleukin (IL)-1β, were assessed. RESULTS EMDs in both carriers significantly increased cell proliferation of both osteoblasts and PDL cells in a similar manner. Both formulations also significantly upregulated the expression of genes encoding BMP2 and TGF-β1 as well as decreased the expression of IL-1β. EMDs in the liquid carrier further retained similar differentiation potential of both osteoblasts and PDL cells by demonstrating increased collagen and osteocalcin gene expression and significantly higher alizarin red staining. CONCLUSIONS The results from the present study indicate that the new formulation of EMDs in a liquid carrier is equally as potent as EMDs in a gel carrier in inducing osteoblast and PDL activity. Future study combining EMDs in a liquid carrier with bone grafting materials is required to further evaluate its potential for combination therapies.

Dynamic Intraligamentary Stabilization (DIS) for treatment of acute anterior cruciate ligament ruptures: case series experience of the first three years.

BACKGROUND In recent years, the scientific discussion has focused on new strategies to enable a torn anterior cruciate ligament (ACL) to heal into mechanically stable scar tissue. Dynamic intraligamentary stabilization (DIS) was first performed in a pilot study of 10 patients. The purpose of the current study was to evaluate whether DIS would lead to similarly sufficient stability and good clinical function in a larger case series. METHODS Acute ACL ruptures were treated by using an internal stabilizer, combined with anatomical repositioning of torn bundles and microfracturing to promote self-healing. Clinical assessment (Tegner, Lysholm, IKDC, and visual analogue scale [VAS] for patient satisfaction scores) and assessment of knee laxity was performed at 3, 6, 12, and 24 months. A one-sample design with a non-inferiority margin was chosen to compare the preoperative and postoperative IKDS and Lysholm scores. RESULTS 278 patients with a 6:4 male to female ratio were included. Average patient age was 31 years. Preoperative mean IKDC, Lysholm, and Tegner scores were 98.8, 99.3, and 5.1 points, respectively. The mean anteroposterior (AP) translation difference from the healthy contralateral knee was 4.7 mm preoperatively. After DIS treatment, the mean 12-month IKDC, Lysholm, and Tegner scores were 93.6, 96.2, and 4.9 points, respectively, and the mean AP translation difference was 2.3 mm. All these outcomes were significantly non-inferior to the preoperative or healthy contralateral values (p < 0.0001). Mean patient satisfaction was 8.8 (VAS 0-10). Eight ACL reruptures occurred and 3 patients reported insufficient subjective stability of the knee at the end of the study period. CONCLUSIONS Anatomical repositioning, along with DIS and microfracturing, leads to clinically stable healing of the torn ACL in the large majority of patients. Most patients exhibited almost normal knee function, reported excellent satisfaction, and were able to return to their previous levels of sporting activity. Moreover, this strategy resulted in stable healing of all sutured menisci, which could lower the rate of osteoarthritic changes in future. The present findings support the discussion of a new paradigm in ACL treatment based on preservation and self-healing of the torn ligament.

Broad Concerns about Nanotechnology Patents: Symptoms and Diagnosis

We discuss the concerns that the patenting activity in the new nanotechnologies could blur the line between what is considered a discovery and what can be considered as an invention. We find that the nature of nanotechnology products, research, and the development agendas in science and engineering fields that include biomimetics pose a challenge to the present practice of including chemicals as eligible patent subject matter. After revisiting the historical development of patent law and noting its divergence from the developments in science and technology, we introduce the distinction between simple and complex machines as these relate to chemistry and nanotechnology. This distinction poses the question of what is the logical category of inventions that fall within patentable subject matter given that patent law was conceived to cover simple machines, not complex ones.