Local induction of regulatory T cells prevents inflammatory bone loss in ligature-induced experimental periodontitis in mice.

Periodontitis (periodontal disease) is a highly prevalent disease, affecting over 65 million adults in the United States alone. Characterized by an overburden of invasive bacteria, gum inflammation and plaque buildup, over time, these symptoms can result in severe loss of gingival tissue attachment, bone resorption and even tooth loss. Although current treatments (local antibiotics and scaling and root planing procedures) target the bacterial dysbiosis, they do not address the underlying inflammatory imbalance in the periodontium. In the healthy steady state, the body naturally combats destructive, imbalanced inflammatory responses through regulatory pathways mediated by cells such as regulatory T cells (Tregs). Consequently, we hypothesized that local enrichment of regulatory lymphocytes (Tregs) could restore local, immunological homeostasis and prevent the main outcome of bone loss. Accordingly, we locally delivered a combination of TGFß, Rapamycin, and IL2 microspheres in a ligature-induced murine periodontitis model. Herein, we have demonstrated this preventative treatment decreases alveolar bone loss, increases the local ratio of Tregs to T effector cells and changes the local microenvironment's expression of inflammatory and regenerative markers. Ultimately, these Treg-inducing microspheres appear promising as a method to improve periodontitis outcomes and may be able to serve as a platform delivery system to treat other inflammatory diseases.

Droplet-based microsystems as novel assessment tools for oral microbial dynamics.

The human microbiome comprises thousands of microbial species that live in and on the body and play critical roles in human health and disease. Recent findings on the interplay among members of the oral microbiome, defined by a personalized set of microorganisms, have elucidated the role of bacteria and yeasts in oral health and diseases including dental caries, halitosis, and periodontal infections. However, the majority of these studies rely on traditional culturing methods which are limited in their ability of replicating the oral microenvironment, and therefore fail to evaluate key microbial interactions in microbiome dynamics. Novel culturing methods have emerged to address this shortcoming. Here, we reviewed the potential of droplet-based microfluidics as an alternative approach for culturing microorganisms and assessing the oral microbiome dynamics. We discussed the state of the art and recent progress in the field of oral microbiology. Although at its infancy, droplet-based microtechnology presents an interesting potential for elucidating oral microbial dynamics and pathophysiology. We highlight how new findings provided by current microfluidic-based methodologies could advance the investigation of the oral microbiome. We anticipate that our work involving the droplet-based microfluidic technique with a semipermeable membrane will lay the foundations for future microbial dynamics studies and further expand the knowledge of the oral microbiome and its implication in oral health.

CCL2 loaded microparticles promote acute patency in silk-based vascular grafts implanted in rat aortae.

Cardiovascular disease is the leading cause of death worldwide, often associated with coronary artery occlusion. A common intervention for arterial blockage utilizes a vascular graft to bypass the diseased artery and restore downstream blood flow; however, current clinical options exhibit high long-term failure rates. Our goal was to develop an off-the-shelf tissue-engineered vascular graft capable of delivering a biological payload based on the monocyte recruitment factor C-C motif chemokine ligand 2 (CCL2) to induce remodeling. Bi-layered silk scaffolds consisting of an inner porous and outer electrospun layer were fabricated using a custom blend of Antherea Assama and Bombyx Mori silk (lyogel). Lyogel silk scaffolds alone (LG), and lyogel silk scaffolds containing microparticles (LGMP) were tested. The microparticles (MPs) were loaded with either CCL2 (LGMP+) or water (LGMP-). Scaffolds were implanted as abdominal aortic interposition grafts in Lewis rats for 1 and 8 weeks. 1-week implants exhibited patency rates of 50% (7/14), 100% (10/10), and 100% (5/5) in the LGMP-, LGMP+, and LG groups, respectively. The significantly higher patency rate for the LGMP+ group compared to the LGMP- group (p = 0.0188) suggests that CCL2 can prevent acute occlusion. Immunostaining of the explants revealed a significantly higher density of macrophages (CD68+ cells) within the outer vs. inner layer of LGMP- and LGMP+ constructs but not in LG constructs. After 8 weeks, there were no significant differences in patency rates between groups. All patent scaffolds at 8 weeks showed signs of remodeling; however, stenosis was observed within the majority of explants. This study demonstrated the successful fabrication of a custom blended silk scaffold functionalized with cell-mimicking microparticles to facilitate controlled delivery of a biological payload improving their in vivo performance. STATEMENT OF SIGNIFICANCE: This study outlines the development of a custom blended silk-based tissue-engineered vascular graft (TEVG) for use in arterial bypass or replacement surgery. A custom mixture of silk was formulated to improve biocompatibility and cellular binding to the tubular scaffold. Many current approaches to TEVGs include cells that encourage graft cellularization and remodeling; however, our technology incorporates a microparticle based delivery platform capable of delivering bioactive molecules that can mimic the function of seeded cells. In this study, we load the TEVGs with microparticles containing a monocyte attractant and demonstrate improved performance in terms of unobstructed blood flow versus blank microparticles. The acellular nature of this technology potentially reduces risk, increases reproducibility, and results in a more cost-effective graft when compared to cell-based options.

Local Sustained Delivery of Anti-IL-17A Antibodies Limits Inflammatory Bone Loss in Murine Experimental Periodontitis.

Periodontal disease (PD) is a chronic destructive inflammatory disease of the tooth-supporting structures that leads to tooth loss at its advanced stages. Although the disease is initiated by a complex organization of oral microorganisms in the form of a plaque biofilm, it is the uncontrolled immune response to periodontal pathogens that fuels periodontal tissue destruction. IL-17A has been identified as a key cytokine in the pathogenesis of PD. Despite its well documented role in host defense against invading pathogens at oral barrier sites, IL-17A-mediated signaling can also lead to a detrimental inflammatory response, causing periodontal bone destruction. In this study, we developed a local sustained delivery system that restrains IL-17A hyperactivity in periodontal tissues by incorporating neutralizing anti-IL-17A Abs in poly(lactic-coglycolic) acid microparticles (MP). This formulation allowed for controlled release of anti-IL-17A in the periodontium of mice with ligature-induced PD. Local delivery of anti-IL-17A MP after murine PD induction inhibited alveolar bone loss and osteoclastic activity. The anti-IL-17A MP formulation also decreased expression of IL-6, an IL-17A target gene known to induce bone resorption in periodontal tissues. This study demonstrates proof of concept that local and sustained release of IL-17A Abs constitutes a promising therapeutic strategy for PD and may be applicable to other osteolytic bone diseases mediated by IL-17A-driven inflammation.

Biorelevant and screening dissolution methods for minocycline hydrochloride microspheres intended for periodontal administration.

Currently, there is no compendial-level method to assess dissolution of particulate systems administered in the periodontal pocket. This work seeks to develop dissolution methods for extended release poly(lactic-co-glycolic acid) (PLGA) microspheres applied in the periodontal pocket. Arestin®, PLGA microspheres containing minocycline hydrochloride (MIN), is indicated for reduction of pocket depth in adult periodontitis. Utilizing Arestin® as a model product, two dissolution methods were developed: a dialysis set-up using USP apparatus 4 and a novel apparatus fabricated to simulate in vivo environment of the periodontal pocket. In the biorelevant method, the microspheres were dispersed in 250 µL of simulated gingival crevicular fluid (sGCF) which was enclosed in a custom-made dialysis enclosure. sGCF was continuously delivered to the device at a biorelevant flow rate and was collected daily for drug content analysis using UPLC. Both methods could discriminate release characteristics of a panel of MIN-loaded PLGA microspheres that differed in composition and process conditions. A mechanistic model was developed, which satisfactorily explained the release profiles observed using both dissolution methods. The developed methods may have the potential to be used as routine quality control tools to ensure batch-to-batch consistency and to support evaluation of bioequivalence for periodontal microspheres.

Design and evaluation of collagen-inspired mineral-hydrogel nanocomposites for bone regeneration.

Bone loss due to trauma and tumors remains a serious clinical concern. Due to limited availability and disease transmission risk with autografts and allografts, calcium phosphate bone fillers and growth factor-based substitute bone grafts are currently used in the clinic. However, substitute grafts lack bone regeneration potential when used without growth factors. When used along with the added growth factors, they lead to unwanted side effects such as uncontrolled bone growth. Collagen-based hydrogel grafts available on the market fail to provide structural guidance to native cells due to high water-solubility and faster degradation. To overcome these limitations, we employed bioinspired material design and fabricated three different hydrogels with structural features similar to native collagen at multiple length-scales. These hydrogels fabricated using polyionic complexation of oppositely charged natural polysaccharides exhibited multi-scale architecture mimicking nanoscale banding pattern, and microscale fibrous structure of native collagen. All three hydrogels promoted biomimetic apatite-like mineral deposition in vitro elucidating crystalline structure on the surface while amorphous calcium phosphate inside the hydrogels resulting in mineral-hydrogel nanocomposites. When evaluated in a non-load bearing critical size mouse calvaria defect model, chitosan - kappa carrageenan mineral-hydrogel nanocomposites enhanced bone regeneration without added growth factors compared to empty defect as well as widely used marketed collagen scaffolds. Histological assessment of the regenerated bone revealed improved healing and tissue remodeling with mineral-hydrogel nanocomposites. Overall, these collagen-inspired mineral-hydrogel nanocomposites showed multi-scale hierarchical structure and can potentially serve as promising bioactive hydrogel to promote bone regeneration. STATEMENT OF SIGNIFICANCE: Hydrogels, especially collagen, are widely used in bone tissue engineering. Collagen fibrils play arguably the most important role during natural bone development. Its multi-scale hierarchical structure to form fibers from fibrils and electrostatic charges enable mineral sequestration, nucleation, and growth. However, bulk collagen hydrogels exhibit limited bone regeneration and are mostly used as carriers for highly potent growth factors such as bone morphogenic protein-2, which increase the risk of uncontrolled bone growth. Thus, there is an unmet clinical need for a collagen-inspired biomaterial that can recreate structural hierarchy, mineral sequestration ability, and stimulate recruitment of host progenitor cells to facilitate bone regeneration. Here, we propose collagen-inspired bioactive mineral-hydrogel nanocomposites as a growth factor-free approach to guide and enhance bone regeneration.

Bottom-Up Self-assembled Hydrogel-Mineral Composites Regenerate Rabbit Ulna Defect without Added Growth Factors.

Hydrogel-based biomaterials have advanced bone tissue engineering approaches in the last decade, through their ability to serve as a carrier for potent growth factor, bone morphogenic protein-2 (BMP-2). However, biophysical properties of hydrogels such as multiscale structural hierarchy and bone extracellular matrix (ECM)-mimetic microarchitecture are underutilized while designing current bone grafts. Incorporation of these properties offers great potential to create a favorable biomimetic microenvironment to harness their regenerative potential. Here, we present our approach to fabricate collagen-inspired bioactive hydrogel scaffolds (referred to as "RegenMatrix") to guide and enhance bone regeneration in a rabbit ulna defect model through the mimicry of multiscale architecture of bone ECM, i.e., native collagen. Specifically, we employed polyelectrolyte complexation to promote bottom-up self-assembly of oppositely charged polysaccharides (chitosan and kappa-carrageenan) at multiple length scales forming fibrils, which further assemble into fibers. The self-assembly and bioinspired scaffold fabrication method resulted in robust cylindrical RegenMatrix with excellent retention of the multiscale architecture and uniform mineral deposition throughout the scaffolds. RegenMatrix, in both nonmineralized and mineralized forms, enhanced bone regeneration in the semiload-bearing ulna defect when compared to the empty defect. RegenMatrix also showed greater histocompatibility without any fibrous tissue formation. Collectively, the RegenMatrix developed in this study has a great potential as a bioactive bone graft without any added growth factors.

Effect of the Periapical "Inflammatory Plug" on Dental Pulp Regeneration: A Histologic In Vivo Study.

INTRODUCTION: In the current study, we investigate the effect of the inflammation occupying the apical foramen-a phenomenon we refer to as "inflammatory plug"-on the regenerative potential of a root canal therapy. METHODS: We performed root canal treatment (RCT) in 12 canine root canals while aseptically instrumenting the apex to a 0.5-mm-wide foramen and obturating the canals with the following materials: collagen sponge, platelet-rich fibrin, and blood clot (no material introduced). RESULTS: We were successful in maintaining the integrity of the periapical tissue in 8 of 12 RCTs. Injury to the periapical tissue occurred during the remaining 4 RCTs, which initiated inflammation accompanied by bone and dentin resorption. Our histologic analyses showed that the resulting inflammatory plug contained abundant M1 macrophages and was associated with an absence of intracanal cellular infiltration. On the contrary, noninflamed samples showed signs of repair, as indicated by the migration of periapical cells throughout the root canal. CONCLUSIONS: We conclude that controlling periapical inflammation is key while attempting to achieve dental pulp regeneration.

Vasoactive Intestinal Peptide Immunoregulatory Role at the Periapex: Associative and Mechanistic Evidences from Human and Experimental Periapical Lesions.

INTRODUCTION: The balance between the host proinflammatory immune response and the counteracting anti-inflammatory and reparative responses supposedly determine the outcome of periapical lesions. In this scenario, the vasoactive intestinal peptide (VIP) may exert a protective role because of its prominent immunoregulatory capacity. In this study, we investigated (in a cause-and-effect manner) the potential involvement of VIP in the development of human and experimental periapical lesions. METHODS: Periapical granulomas (n = 124) and control samples (n = 48) were comparatively assessed for VIP and multiple immunologic/activity marker expression through real-time polymerase chain reaction. Experimental periapical lesions (C57Bl/6 wild-type mice) were evaluated regarding endogenous VIP expression correlation with lesion development and the effect of recombinant VIP therapy in lesion outcome. CCR4KO and IL4KO strains and anti-glucocorticoid-induced TNFR-related protein inhibition were used to test the involvement of Treg and Th2 cells in VIP-mediated effects. RESULTS: VIP expression was more prevalent in periapical granulomas than in controls, presenting a positive association with immunoregulatory factors and an inverse association/correlation with proinflammatory mediators and the receptor activator of nuclear factor kappa B ligand/osteoprotegerin ratio. Endogenous VIP expression up-regulation was temporally associated with lesion immunoregulation and a decline of bone loss. VIP therapy in mice prompted the arrest of lesion development, being associated with an anti-inflammatory and proreparative response that limits the proinflammatory, Th1, Th17, and osteoclastogenic response in the periapex. The VIP protective effect was dependent of Treg migration and activity and independent of interleukin 4. CONCLUSIONS: Our results show that VIP overexpression in human and experimental periapical lesions is associated with lesion inactivity and that VIP therapy results in the attenuation of experimental lesion progression associated with the immunosuppressive response involving Treg cells.

Controlling magnesium corrosion and degradation-regulating mineralization using matrix GLA protein.

Magnesium (Mg) alloys are embraced for their biodegradability and biocompatibility. However, Mg degrades spontaneously in the biological environment in vivo and in vitro, triggering deposition of calcium phosphate on the metal. Upon complete metal absorption, minerals remain in the tissue, which could lead to pathogenic calcification. Hence, our aims are to test the feasibility of matrix GLA protein (MGP) to locally inhibit Mg mineralization that is induced by metal alloy degradation. MGP is a small secretory protein that has been shown to inhibit soft tissue calcification. We exposed Mg to MGP, stably transfected into mammalian cells. Results showed that less calcium and phosphorous deposition on the Mg surface when MGP was present relative to when it was not. In the in vivo mouse intramuscular model conducted for 4 and 6 weeks, Mg rods were embedded in collagen scaffolds, seeded with cells overexpressing MGP. Microtomography, electron dispersive x-ray spectroscopy, and histology assessments revealed lower deposited mineral volume around Mg rods from the MGP group. Compared to other groups, higher volume loss after implantation was observed from the MGP group at both time points, indicating a higher corrosion rate without the protective mineral layer. This study is the first to our knowledge to demonstrate that local exposure to a biomolecule, such as MGP, can modulate the corrosion of Mg-based implants. These findings may have important implications for the future design of endovascular stents and orthopedic devices.

Prx1 Expressing Cells Are Required for Periodontal Regeneration of the Mouse Incisor.

Previous studies have shown that post-natal skeletal stem cells expressing Paired-related homeobox 1 (PRX1 or PRRX1) are present in the periosteum of long bones where they contribute to post-natal bone development and regeneration. Our group also identified post-natal PRX1 expressing cells (pnPRX1+ cells) in mouse calvarial synarthroses (sutures) and showed that these cells are required for calvarial bone regeneration. Since calvarial synarthroses are similar to dentoalveolar gomphosis (periodontium) and since there is no information available on the presence or function of pnPRX1+ cells in the periodontium, the present study aimed at identifying and characterizing pnPRX1+ cells within the mouse periodontium and assess their contribution to periodontal development and regeneration. Here we demonstrated that pnPRX1+ cells are present within the periodontal ligament (PDL) of the mouse molars and of the continuously regenerating mouse incisor. By means of diphtheria toxin (DTA)-mediated conditional ablation of pnPRX1+ cells, we show that pnPRX1+ cells contribute to post-natal periodontal development of the molars and the incisor, as ablation of pnPRX1+ cells in 3-days old mice resulted in a significant enlargement of the PDL space after 18 days. The contribution of pnPRX1+ cells to periodontal regeneration was assessed by developing a novel non-critical size periodontal defect model. Outcomes showed that DTA-mediated post-natal ablation of pnPRX1+ cells results in lack of regeneration in periodontal non-critical size defects in the regeneration competent mouse incisors. Importantly, gene expression analysis of these cells shows a profile typical of quiescent cells, while gene expression analysis of human samples of periodontal stem cells (PDLSC) confirmed that Prx1 is highly expressed in human periodontium. In conclusion, pnPRX1+ cells are present within the continuously regenerating PDL of the mouse incisor, and at such location they contribute to post-natal periodontal development and regeneration. Since this study further reports the presence of PRX1 expressing cells within human periodontal ligament, we suggest that studying the mouse periodontal pnPRX1+ cells may provide significant information for the development of novel and more effective periodontal regenerative therapies in humans.

The role of magnesium ions in bone regeneration involves the canonical Wnt signaling pathway.

Magnesium (Mg)-based implants have become of interest to both academia and the medical industry. The attraction largely is due to Mg's biodegradability and ability to enhance bone healing and formation. However, the underlying mechanism of how Mg regulates osteogenesis is still unclear. Based on our previous in vivo and molecular signaling work demonstrating the osteogenic effect of Mg, the current study aims to extend this work at the molecular level especially that we also observed and quantified mineral deposits in the bone marrow space in a rabbit ulna fracture model with Mg plates and screws. Histological analysis and quantitative results of micro-CT showed mineralized deposition and a significant increase in bone volume at 8 weeks and 16 weeks post-operative. These in vivo results led us to focus on studying the effect of Mg2+ on human bone marrow stromal cells (hBMSCs). The data presented in this manuscript demonstrate the activation of the canonical Wnt signaling pathway in hBMSCs when treated with 10 mM Mg2+. With additional Mg2+ present, the protein expression of active ß-catenin was significantly increased to a level similar to that of the positive control. Immunocytochemistry and the increased expression of LEF1 and Dkk1, downstream target genes that are controlled directly by active ß-catenin, demonstrated the protein translocation and the activation of transcription. Taken together, these data suggest that Mg2+ induces an osteogenic effect in the bone marrow space by activating the canonical Wnt signaling pathway, which in turn causes BMSCs to differentiate toward the osteoblast lineage. STATEMENT OF SIGNIFICANCE: Magnesium (Mg)-based alloys are being studied to be used in the field of implantable medical devices due to its natural biodegradability and the potential ability to promote bone regeneration. Despite many in vivo studies that demonstrated an increased new bone growth by implanting Mg-based devices, the underlying mechanism of this effect is still unclear. In order to safely use Mg-based implants on human and better control the osteogenic effect, it is necessary to understand the corresponding cellular response in the targeted area. The present study provides the rationale to study Mg ions on bone marrow stromal cells and shows the activation of canonical Wnt signaling pathway that promotes osteogenesis by in vivo and in vitro approaches.

Sterilization and Biologic Monitoring in Private Dental Clinics in Lebanon.

AIM: The aim of the present study was to evaluate sterilization practices and effectiveness in the Lebanese private dental sector and identify potential factors contributing to sterilization failure. MATERIALS AND METHODS: A 13-item questionnaire consisting of four demographic/professional questions and nine questions related to sterilization practices along with self-contained biologic indicators (SCBIs) were delivered to a representative sample of Lebanese private offices. Univariate statistics and bivariate analyses were performed to compare sterilization failure rates according to demographic, professional, and sterilization-related conditions. RESULTS: Out of the 560 dentists contacted, 205 dentists returned the completed questionnaires and undamaged processed SCBIs. The tested autoclaves (n = 134) were mostly dynamic air removal (69.4%) and had a mean age of 10.5 ± 6.9 years. The dry heat ovens (n = 71) were all static air and had 12.9 ± 8.1 years. The dental assistants performed the routine sterilization procedures in nearly 62% of the practices and sterilization cycles were run 4 to 6 times per week in 75% of the offices. Correct temperature/time ratios were applied in 97% of the autoclaves and 80.3% of the ovens. Few dental practices reported having preventive maintenance (17.9% for the autoclaves and 14.1% for the ovens). Routine monitoring of sterilizer efficacy was infrequently performed and was mostly conducted using physical indicators. Sterilization failure rate was higher for the ovens (16.9%) than for the autoclaves (7.5%). Incorrect temperature/time ratio was the main significant factor associated with sterilization failures. CONCLUSION: The present study demonstrated a relatively high rate of sterilization failures in the Lebanese private dental sector and identified the human error in setting sterilization cycle parameters as the predominant cause of failure. These findings should prompt actions toward increasing knowledge of the sterilization processes and their monitoring among dental professionals and improving the quality control of sterilization through collaborative efforts among health authorities, dental schools, and associations. CLINICAL SIGNIFICANCE: This study presents the first published data relative to sterilization practices and effectiveness in private Lebanese dental offices and provides a rationale to implement biologic monitoring protocols in Lebanon as long practiced in developed countries.

In vivo quantification of hydrogen gas concentration in bone marrow surrounding magnesium fracture fixation hardware using an electrochemical hydrogen gas sensor.

Magnesium (Mg) medical devices are currently being marketed for orthopedic applications and have a complex degradation process which includes the evolution of hydrogen gas (H2). The effect of H2 exposure on relevant cell types has not been studied; and the concentration surrounding degrading Mg devices has not been quantified to enable such mechanistic studies. A simple and effective method to measure the concentration of H2 in varying microenvironments surrounding Mg implants is the first step to understanding the biological impact of H2 on these cells. Here, the in vivo measurement of H2 surrounding fracture fixation devices implanted in vivo is demonstrated. An electrochemical H2 microsensor detected increased levels of H2 at three anatomical sites with a response time of about 30 s. The sensor showed the H2 concentration in the bone marrow at 1 week post-implantation (1460 ±â€¯320 µM) to be much higher than measured in the subcutaneous tissue (550 ±â€¯210 µM) and at the skin surface (120 ±â€¯50 µM). Additionally, the H2 concentrations measured in the bone marrow exceeded the concentration in a H2 saturated water solution (∼800 µM). These results suggest that H2 emanating from Mg implants in bone during degradation pass through the bone marrow and become at least partially trapped because of slow permeation through the bone. This study is the first to identify H2 concentrations in the bone marrow environment and will enable in vitro experiments to be executed at clinically relevant H2 concentrations to explore possible biological effects of H2 exposure. STATEMENT OF SIGNIFICANCE: An electrochemical H2 sensor was used to monitor the degradation of a Mg fracture fixation system in a lapine ulna fracture model. Interestingly, the H2 concentration in the bone marrow is 82% higher than H2 saturated water solution. This suggests H2 generated in situ is trapped in the bone marrow and bone is less permeable than the surrounding tissues. The detectable H2 at the rabbit skin also demonstrates a H2 sensor's ability to monitor the degradation process under thin layers of tissue. H2 sensing shows promise as a tool for monitoring the degradation of Mg alloy in vivo and creating in vitro test beds to more mechanistically evaluate the effects of varying H2 concentrations on cell types relevant to osteogenesis.

Infection Control Measures in Private Dental Clinics in Lebanon.

PURPOSE: Evaluate infection control knowledge, attitude, and practice in Lebanese private dental clinics. MATERIALS AND METHODS: A survey including 46 questions related to routine safety procedures was sent to 1150 Lebanese dentists between July 1st and 2nd, 2015. The study sample was selected from the database of registered dentists based on a proportional random sampling ensuring equitable representation of the 5 geographic regions of Lebanon. A subset of 29 questions was used to generate an overall score of compliance (excellent, good, fair, and poor). Comparisons according to gender, type, region, and years of practice were performed. RESULTS: 417 dentists returned the completed questionnaires. 96% expressed concern about infection transmission, 90.6% were vaccinated against Hepatitis B, and 61.8% asked routinely about patients medical history. Only 43% used protective eyewear. Although most dentists (65%) used autoclaves, dry heat was still used. Significant correlations were found between gender and use of personal protective equipment. Less compliance was shown by clinicians with fewer years of experience. In the overall compliance questionnaire, the mean percentage of correct answers was roughly 54% with <5% of the practitioners scoring "excellent." Conclusions. The study found inadequacy of compliance in private Lebanese dental clinics necessitating improved educational training and sustained monitoring by regulatory bodies.

Characterization of the Protective Role of Regulatory T Cells in Experimental Periapical Lesion Development and Their Chemoattraction Manipulation as a Therapeutic Tool.

INTRODUCTION: The pathogenesis of periapical lesions is determined by the balance between host proinflammatory immune response and counteracting anti-inflammatory and reparative responses, which include regulatory T cells (Tregs) as potential immunoregulatory agents. In this study, we investigated (in a cause-and-effect manner) the involvement of CCL22-CCR4 axis in Treg migration to the periapical area and the role of Tregs in the determination of outcomes in periapical lesions. METHODS: Periapical lesions were induced in C57Bl/6 (wild-type) and CCR4KO mice (pulp exposure and bacterial inoculation) and treated with anti-glucocorticoid-induced TNF receptor family regulated gene to inhibit Treg function or alternatively with CCL22-releasing, polylactic-glycolic acid particles to induce site-specific migration of Tregs. After treatment, lesions were analyzed for Treg influx and phenotype, overall periapical bone loss, and inflammatory/immunologic and wound healing marker expression (analyzed by real-time polymerase chain reaction array). RESULTS: Treg inhibition by anti-glucocorticoid-induced TNF receptor family regulated gene or CCR4 depletion results in a significant increase in periapical lesion severity, associated with upregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with decreased Treg and healing marker expression. The local release of CCL22 in the root canal system resulted in the promotion of Treg migration in a CCR4-dependent manner, leading to the arrest of periapical lesion progression, associated with downregulation of proinflammatory, T-helper 1, T-helper 17, and tissue destruction markers in parallel with increased Treg and healing marker expression. CONCLUSIONS: Because the natural and CCL22-induced Treg migration switches active lesion into inactivity phenotype, Treg chemoattractant may be a promising strategy for the clinical management of periapical lesions.

Aquaporin 5 Interacts with Fluoride and Possibly Protects against Caries.

Aquaporins (AQP) are water channel proteins and the genes coding for AQP2, AQP5, and AQP6 are clustered in 12q13. Since AQP5 is expressed in serous acinar cells of salivary glands, we investigated its involvement in caries. DNA samples from 1,383 individuals from six groups were studied. Genotypes of eight single nucleotide polymorphisms covering the aquaporin locus were tested for association with caries experience. Interaction with genes involved in enamel formation was tested. The association between enamel microhardness at baseline, after creation of artificial caries lesion, and after exposure to fluoride and the genetic markers in AQP5 was tested. Finally, AQP5 expression in human whole saliva, after exposure to fluoride in a mammary gland cell line, which is known to express AQP5, and in Wistar rats was also verified. Nominal associations were found between caries experience and markers in the AQP5 locus. Since these associations suggested that AQP5 may be inhibited by levels of fluoride in the drinking water that cause fluorosis, we showed that fluoride levels above optimal levels change AQP5 expression in humans, cell lines, and rats. We have shown that AQP5 is involved in the pathogenesis of caries and likely interacts with fluoride.

An in vivo model to assess magnesium alloys and their biological effect on human bone marrow stromal cells.

Magnesium (Mg) alloys have many unique qualities which make them ideal candidates for bone fixation devices, including biocompatibility and degradation in vivo. Despite a rise in Mg alloy production and research, there remains no standardized system to assess their degradation or biological effect on human stem cells in vivo. In this study, we developed a novel in vivo model to assess Mg alloys for craniofacial and orthopedic applications. Our model consists of a collagen sponge seeded with human bone marrow stromal cells (hBMSCs) around a central Mg alloy rod. These scaffolds were implanted subcutaneously in mice and analyzed after eight weeks. Alloy degradation and biological effect were determined by microcomputed tomography (microCT), histological staining, and immunohistochemistry (IHC). MicroCT showed greater volume loss for pure Mg compared to AZ31 after eight weeks in vivo. Histological analysis showed that hBMSCs were retained around the Mg implants after 8 weeks. Furthermore, immunohistochemistry showed the expression of dentin matrix protein 1 and osteopontin around both pure Mg and AZ31 with implanted hBMSCs. In addition, histological sections showed a thin mineral layer around all degrading alloys at the alloy-tissue interface. In conclusion, our data show that degrading pure Mg and AZ31 implants are cytocompatible and do not inhibit the osteogenic property of hBMSCs in vivo. These results demonstrate that this model can be used to efficiently assess the biological effect of corroding Mg alloys in vivo. Importantly, this model may be modified to accommodate additional cell types and clinical applications. STATEMENT OF SIGNIFICANCE: Magnesium (Mg) alloys have been investigated as ideal candidates for bone fixation devices due to high biocompatibility and degradation in vivo, and there is a growing need of establishing an efficient in vivo material screening system. In this study, we assessed degradation rate and biological effect of Mg alloys by transplanting Mg alloy rod with human bone marrow stromal cells seeded on collagen sponge subcutaneously in mice. After 8 weeks, samples were analyzed by microcomputed tomography and histological staining. Our data show that degrading Mg alloys are cytocompatible and do not inhibit the osteogenic property of hBMSCs in vivo. These results demonstrate that this model can be used to efficiently assess the biological effect of corroding Mg alloys in vivo.

In vivo study of magnesium plate and screw degradation and bone fracture healing.

Each year, millions of Americans suffer bone fractures, often requiring internal fixation. Current devices, like plates and screws, are made with permanent metals or resorbable polymers. Permanent metals provide strength and biocompatibility, but cause long-term complications and may require removal. Resorbable polymers reduce long-term complications, but are unsuitable for many load-bearing applications. To mitigate complications, degradable magnesium (Mg) alloys are being developed for craniofacial and orthopedic applications. Their combination of strength and degradation make them ideal for bone fixation. Previously, we conducted a pilot study comparing Mg and titanium devices with a rabbit ulna fracture model. We observed Mg device degradation, with uninhibited healing. Interestingly, we observed bone formation around degrading Mg, but not titanium, devices. These results highlighted the potential for these fixation devices. To better assess their efficacy, we conducted a more thorough study assessing 99.9% Mg devices in a similar rabbit ulna fracture model. Device degradation, fracture healing, and bone formation were evaluated using microcomputed tomography, histology and biomechanical tests. We observed device degradation throughout, and calculated a corrosion rate of 0.40±0.04mm/year after 8 weeks. In addition, we observed fracture healing by 8 weeks, and maturation after 16 weeks. In accordance with our pilot study, we observed bone formation surrounding Mg devices, with complete overgrowth by 16 weeks. Bend tests revealed no difference in flexural load of healed ulnae with Mg devices compared to intact ulnae. These data suggest that Mg devices provide stabilization to facilitate healing, while degrading and stimulating new bone formation.