Effects of radiopaque double antibiotic pastes on the proliferation, alkaline phosphatase activity and mineral deposition of dental pulp stem cells.

OBJECTIVE: The aim of this study was to investigate the effects of two radiopaque agents, barium sulfate (BaSO4) or zirconium oxide (ZrO2) in double antibiotic paste (DAP), on the proliferation and mineral deposition of human dental pulp stem cells (DPSC). MATERIALS AND METHODS: Radiopaque antimicrobial medicaments composed of methylcellulose (MC) thickening polymer with BaSO4 or ZrO2 and either 1 or 5 mg/mL DAP (equal portions of metronidazole and ciprofloxacin) were used to investigate DPSC proliferation after 3 days, and alkaline phosphatase (ALP) activity and mineral deposition after 7 and 14 days. Radiopaque agents without DAP and Ca(OH)2 were used as controls. RESULTS: MC-BaSO4 DAP and MC-ZrO2 DAP at 1 or 5 mg/mL had no adverse effect on DPSC proliferation, compared to the media and MC controls. MC-ZrO2 (DAP-free) greatly increased ALP activity after 7 days. DPSC mineral deposition was modestly reduced at 7 days by MC-BaSO4 DAP and MC-ZrO2 DAP, but not by DAP-free radiopaque agents, and was most reduced by 5 mg/mL DAP in the 14-day cultures. CONCLUSIONS: MC-BaSO4 or MC-ZrO2 medicaments containing up to 5 mg/mL of DAP supported the proliferation and early osteogenic differentiation of DPSC. Low DAP concentrations and short culture times led to more favorable effects on ALP activity and mineral deposition by DPSC. The findings suggest that radiopaque agents added for the purpose of detecting whether medicaments occupy the full extent of the root canal may have clinical applications. Radiopaque antibiotic medicaments containing low DAP concentrations may be an alternative to Ca(OH)2 for regenerative endodontic procedures.

The antimicrobial properties, cytotoxicity, and differentiation potential of double antibiotic intracanal medicaments loaded into hydrogel system.

OBJECTIVE: This study evaluated the antimicrobial properties, cytotoxicity, and mineralization potential of methylcellulose hydrogels loaded with low concentrations of double antibiotic pastes (DAP). MATERIALS AND METHODS: The direct and residual antibacterial effects of 1, 5, and 10 mg/mL of DAP loaded into hydrogels as well as calcium hydroxide (Ca(OH)2) were tested against single-species biofilms of Enterococcus faecalis and dual-species biofilms (Enterococcus faecalis and Prevotella intermedia). The effects of DAP hydrogels on proliferation and mineralization of dental pulp stem cells (DPSC) were tested using MTT assays, alkaline phosphate activity (ALP), and alizarin red staining. Fisher's exact tests, Wilcoxon rank sum tests, and one-way ANOVA were used for statistical analyses (α = 0.05). RESULTS: All tested concentrations of DAP hydrogels as well as Ca(OH)2 demonstrated significant direct antibacterial effects against single- and dual-species biofilms. However, only 5 and 10 mg/mL of DAP hydrogels exhibited significant residual antibacterial effects against both types of tested biofilms. Only 1 mg/mL of DAP hydrogels did not have significant negative effects on DPSC viability, ALP activity, and mineralization nodule formation. However, 5 and 10 mg/mL of DAP hydrogels caused significant negative effects on cytotoxicity and mineralization nodule formation of DPSC. CONCLUSIONS: Hydrogels containing 1 mg/mL DAP offered significant direct antibacterial effects against single- and dual-species biofilms without causing significant negative effects on viability, ALP activity, and mineralization nodule formation of DPSC. CLINICAL RELEVANCE: The methylcellulose-based hydrogel proposed in this study can be used clinically as a biocompatible system to deliver controlled low concentrations of DAP.

Pyk2 deficiency potentiates osteoblast differentiation and mineralizing activity in response to estrogen or raloxifene.

Bone remodeling is controlled by the actions of bone-degrading osteoclasts and bone-forming osteoblasts (OBs). Aging and loss of estrogen after menopause affects bone mass and quality. Estrogen therapy, including selective estrogen receptor modulators (SERMs), can prevent bone loss and increase bone mineral density in post-menopausal women. Although investigations of the effects of estrogen on osteoclast activity are well advanced, the mechanism of action of estrogen on OBs is still unclear. The proline-rich tyrosine kinase 2 (Pyk2) is important for bone formation and female mice lacking Pyk2 (Pyk2-KO) exhibit elevated bone mass, increased bone formation rate and reduced osteoclast activity. Therefore, in the current study, we examined the role of estrogen signaling on the mechanism of action of Pyk2 in OBs. As expected, Pyk2-KO OBs showed significantly higher proliferation, matrix formation, and mineralization than WT OBs. In addition we found that Pyk2-KO OBs cultured in the presence of either 17ß-estradiol (E2) or raloxifene, a SERM used for the treatment of post-menopausal osteoporosis, showed a further robust increase in alkaline phosphatase (ALP) activity and mineralization. We examined the possible mechanism of action and found that Pyk2 deletion promotes the proteasome-mediated degradation of estrogen receptor α (ERα), but not estrogen receptor ß (ERß). As a consequence, E2 signaling via ERß was enhanced in Pyk2-KO OBs. In addition, we found that Pyk2 deletion and E2 stimulation had an additive effect on ERK phosphorylation, which is known to stimulate cell differentiation and survival. Our findings suggest that in the absence of Pyk2, estrogen exerts an osteogenic effect on OBs through altered ERα and ERß signaling. Thus, targeting Pyk2, in combination with estrogen or raloxifene, may be a novel strategy for the prevention and/or treatment of bone loss diseases.

ASSESSMENT OF RETINAL BLOOD FLOW IN DIABETIC RETINOPATHY USING DOPPLER FOURIER-DOMAIN OPTICAL COHERENCE TOMOGRAPHY.

PURPOSE: To evaluate retinal blood flow measurements in normal eyes and eyes with varying levels of diabetic retinopathy (DR) using Doppler Fourier-domain optical coherence tomography (FD-OCT). METHODS: Twenty-two eyes of 19 subjects, 10 with severe nonproliferative DR (NPDR) and 12 with proliferative DR (PDR), were compared with 44 eyes of 40 healthy control subjects. All eyes were scanned by RTvue FD-OCT. Color disk photographs and cube/volume scans of the optic nerve head were obtained. Doppler OCT scans and accessory imaging data were imported into Doppler OCT of Retinal Circulation grading software to calculate TRBF and vascular parameters (e.g., venous and arterial cross-sectional area). Measurements were compared between cases and controls using independent t-tests. RESULTS: Mean TRBF was 44.98 ± 9.80 (range: 30.18-64.58) µL/minute for normal eyes, 35.80 ± 10.48 (range: 20.69-49.56) µL/minute for eyes with severe NPDR, and 34.79 ± 10.61 (range: 16.77-48.9) µL/minute for eyes with PDR. Mean TRBF was significantly lower in eyes with severe NPDR (P = 0.01) and PDR (P = 0.003) than in normal eyes. CONCLUSION: Total retinal blood flow was significantly lower in eyes with severe NPDR and PDR compared with normal eyes. Retinal blood flow determined by Doppler OCT may be a useful parameter for evaluating patients with DR.

A Highly Efficacious Herpes Simplex Virus 1 Vaccine Blocks Viral Pathogenesis and Prevents Corneal Immunopathology via Humoral Immunity.

UNLABELLED: Correlates of immunologic protection requisite for an efficacious herpes simplex virus 1 (HSV-1) vaccine remain unclear with respect to viral pathogenesis and clinical disease. In the present study, mice were vaccinated with a novel avirulent, live attenuated virus (0ΔNLS) or an adjuvanted glycoprotein D subunit (gD-2) similar to that used in several human clinical trials. Mice vaccinated with 0ΔNLS showed superior protection against early viral replication, neuroinvasion, latency, and mortality compared to that of gD-2-vaccinated or naive mice following ocular challenge with a neurovirulent clinical isolate of HSV-1. Moreover, 0ΔNLS-vaccinated mice exhibited protection against ocular immunopathology and maintained corneal mechanosensory function. Vaccinated mice also showed suppressed T cell activation in the draining lymph nodes following challenge. Vaccine efficacy correlated with serum neutralizing antibody titers. Humoral immunity was identified as the correlate of protection against corneal neovascularization, HSV-1 shedding, and latency through passive immunization. Overall, 0ΔNLS affords remarkable protection against HSV-1-associated ocular sequelae by impeding viral replication, dissemination, and establishment of latency. IMPORTANCE: HSV-1 manifests in a variety of clinical presentations ranging from a rather benign "cold sore" to more severe forms of infection, including necrotizing stromal keratitis and herpes simplex encephalitis. The present study was undertaken to evaluate a novel vaccine to ocular HSV-1 infection not only for resistance to viral replication and spread but also for maintenance of the visual axis. The results underscore the necessity to reconsider strategies that utilize attenuated live virus as opposed to subunit vaccines against ocular HSV-1 infection.