Low-dose xenogeneic mesenchymal stem cells target canine osteoarthritis through systemic immunomodulation and homing.

BACKGROUND: As current therapies for canine osteoarthritis (OA) provide mainly symptomatic improvement and fail to address the complex pathology of the disease, mesenchymal stem cells (MSCs) offer a promising biological approach to address both aspects of OA through their immunomodulatory properties. METHODS: This study aimed to investigate the safety and efficacy of xenogeneic MSCs in dogs with OA at different dose levels after intravenous injection. OA was surgically induced in the right stifle joint. Thirty-two male and female dogs were divided into three treatment groups and a control group. Regular general physical examinations; lameness, joint, radiographic, and animal caretaker assessments; pressure plate analyses; and blood analyses were performed over 42 days. At study end, joint tissues were evaluated regarding gross pathology, histopathology, and immunohistochemistry. In a follow-up study, the biodistribution of intravenously injected 99mTc-labeled equine peripheral blood-derived MSCs was evaluated over 24h in three dogs after the cruciate ligament section. RESULTS: The dose determination study showed the systemic administration of ePB-MSCs in a canine OA model resulted in an analgesic, anti-inflammatory, and joint tissue protective effect associated with improved clinical signs and improved cartilage structure, as well as a good safety profile. Furthermore, a clear dose effect was found with 0.3 × 106 ePB-MSCs as the most effective dose. In addition, this treatment was demonstrated to home specifically towards the injury zone in a biodistribution study. CONCLUSION: This model-based study is the first to confirm the efficacy and safety of systemically administered xenogeneic MSCs in dogs with OA. The systemic administration of a low dose of xenogeneic MSCs could offer a widely accessible, safe, and efficacious treatment to address the complex pathology of canine OA and potentially slow down the disease progression by its joint tissue protective effect.

Equine allogeneic tenogenic primed mesenchymal stem cells: A clinical field study in horses suffering from naturally occurring superficial digital flexor tendon and suspensory ligament injuries.

BACKGROUND: Mesenchymal stem cells are an innovative therapeutic for various equine orthopaedic diseases, including soft tissue injuries. OBJECTIVES: To evaluate the safety and efficacy of tenogenic primed equine allogeneic peripheral blood-derived mesenchymal stem cells (tpMSCs) in horses with naturally occurring superficial digital flexor tendon (SDFT) and suspensory ligament (SL) injuries. STUDY DESIGN: Multicentre, blinded, randomised, placebo-controlled clinical trial. METHODS: One hundred client-owned horses with SDFT and SL injuries were randomised to receive an intralesional tpMSC (66) or saline (34) injection. Clinical and ultrasonographic evaluation was performed before treatment and on Days 56 ± 3 and 112 ± 3 after treatment. Long-term data on re-injury was collected up to 2 years after treatment. RESULTS: Significantly more tpMSC-treated horses achieved improvement in fibre alignment score (FAS) (100% vs. 54.5%, p < 0.001) and echogenicity (97.0% vs. 57.6%, p < 0.001) on Day 112 ± 3, and their lesion size decreased significantly (-27.6 ± 25.91 vs. -4.6 ± 26.64 mm2 , p < 0.001) compared to the placebo group. A FAS = 0 was achieved in 65% of tpMSC-treated horses, as compared to 9% of placebo-treated horses at Day 112 ± 3. The attending veterinarians reported no re-injury in 41 of 53 tpMSC and in 2 of 26 saline-treated horses available for long-term follow-up (p < 0.001). MAIN LIMITATIONS: As this study consisted of client-owned horses, no samples for histology were collected. Long-term follow-up was only available for a subset of enrolled horses. CONCLUSIONS: The intralesional administration of tpMSCs was safe and improved the quality of healing and long-term outcomes in sports horses with naturally occurring SDFT and suspensory injuries.

Breast cancer morbidity and mortality in rural Ethiopia: data from 788 verbal autopsies.

INTRODUCTION: In Ethiopia, breast cancer is the leading cause of cancer among women. Little is known about the duration of disease and symptoms of patients who died from breast cancer in rural Ethiopia. The objective of this study was to assess breast cancer mortality with a particular focus on the self-reported duration of illness including suffering of symptoms, and need for medical care. METHODS: The cause of death was determined among randomly selected Ethiopian women residing in western Ethiopia. A modified standard verbal autopsy (VA) questionnaire was completed by women whose sisters had died. The questionnaires were reviewed by two independent local physicians to assign a cause of death. We analyzed pattern of cause of deaths, duration of suffering, symptoms, and treatment received. RESULT: In our study, the age at death was very similar to other population-based data from Ethiopia. We found 32% of 788 deaths were caused by communicable diseases, 12.1% by neoplasms, and 9.4% by pregnancy/maternal mortality. Breast cancer was the second leading neoplasm, responsible for 21 (2.7%) of all deaths (95% CI 1.5-3.7%), and was among the top five causes of non-communicable deaths. The median age of breast cancer death was 37 years, younger than for other causes of death. The median duration of illness with breast cancer was around 1 year. This was substantially more compared to the duration of infectious diseases, but less than the duration of reproductive neoplasms, diabetes, and epilepsy. DISCUSSION/CONCLUSION: Breast cancer deaths are common causes of death in women of rural Ethiopia. When assessing the total duration of illness according to specific causes of death, breast and other cancers accounted for a large share of the burden. This has practical implications and highlights the need for palliative care for cancer patients. Substantial efforts are necessary to improve early detection and treatment for breast cancer to reduce premature death in women.

Pre-incubation of corneal donor tissue with sCD83 improves graft survival via the induction of alternatively activated macrophages and tolerogenic dendritic cells.

Immune responses reflect a complex interplay of cellular and extracellular components which define the microenvironment of a tissue. Therefore, factors that locally influence the microenvironment and re-establish tolerance might be beneficial to mitigate immune-mediated reactions, including the rejection of a transplant. In this study, we demonstrate that pre-incubation of donor tissue with the immune modulator soluble CD83 (sCD83) significantly improves graft survival using a high-risk corneal transplantation model. The induction of tolerogenic mechanisms in graft recipients was achieved by a significant upregulation of Tgfb, Foxp3, Il27, and Il10 in the transplant and an increase of regulatory dendritic cells (DCs), macrophages (Mφ), and T cells (Tregs) in eye-draining lymph nodes. The presence of sCD83 during in vitro DC and Mφ generation directed these cells toward a tolerogenic phenotype leading to reduced proliferation-stimulating activity in MLRs. Mechanistically, sCD83 induced a tolerogenic Mφ and DC phenotype, which favors Treg induction and significantly increased transplant survival after adoptive cell transfer. Conclusively, pre-incubation of corneal grafts with sCD83 significantly prolongs graft survival by modulating recipient Mφ and DCs toward tolerance and thereby establishing a tolerogenic microenvironment. This functional strategy of donor graft pre-treatment paves the way for new therapeutic options in the field of transplantation.

Topical VEGF-C/D Inhibition Prevents Lymphatic Vessel Ingrowth into Cornea but Does Not Improve Corneal Graft Survival.

Vascular endothelial growth factor-C/D (VEGF-C/D) regulates lymphangiogenesis. Ingrowth of lymphatic vessels is negatively associated with corneal transplantation success. In this study, we therefore analyzed the effect local blockade of VEGF-C/D has on inflamed corneas. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation. Mice were treated with a VEGF-C/D trap prior to transplantation. Topical inhibition of VEGF-C/D significantly reduced lymphatic vessel ingrowth, but increased Macrophage numbers in the cornea. Furthermore, corneal transplantation success was not improved by the topical application of the compound. This study demonstrates that local VEGF-C/D inhibition is insufficient to increases corneal transplantation success, likely due to interaction with immune cells.

The Multicomponent, Multitarget Therapy SUC in Cats with Chronic Kidney Disease: A Multicenter, Prospective, Observational, Nonrandomized Cohort Study.

BACKGROUND: We compared the natural multicomponent, multitarget therapy SUC (Solidago compositum ad us. vet., Ubichinon compositum and Coenzyme compositum, Heel GmbH, Baden-Baden, Germany) to the well-known angiotensin-converting enzyme inhibitor benazepril in a prospective, observational, nonrandomized, two-arm cohort study of cats with chronic kidney disease (CKD). The objective was to assess the tolerability and the effectiveness of SUC in cats with CKD. MATERIAL AND METHODS: One hundred thirty-six cats were screened for CKD, and 70 cats were eligible for the study. Thirty-three cats were assigned to the SUC treatment, and 35 cats received benazepril. All cats were diagnosed with CKD. The follow-up period was 168 days. Response was assessed as an improved or stable serum creatinine from baseline to the end of the study. Additionally, a clinical summary score, as measure of quality of life, was evaluated. RESULTS: Serum creatinine remained close to baseline in both study groups with slightly improved values in the SUC group. The clinical summary score improved significantly in the SUC group on days 3, 7, 28, 56 and 112, but not on day 168. CONCLUSIONS: Within the limitations of the study, the results carry implications for the usefulness of SUC as an interesting new treatment option for feline CKD. The results indicate that SUC might be more effective if given at least twice weekly.

Bevacizumab Induces Upregulation of Keratin 3 and VEGFA in Human Limbal Epithelial Cells in Vitro.

Topical application of vascular endothelial growth factor A (VEGFA) inhibitors including Bevacizumab is used for antiangiogenic therapy at the ocular surface. While clinical studies have suggested that this approach is well-tolerated, the effect of the drug on limbal epithelial stem cells has not been studied. In this study, the effect of Bevacizumab on phenotype and functionality of putative limbal epithelial stem cells (SC) was investigated. The effect of Bevacizumab on human limbal epithelial cells was assessed in terms of metabolic activity and scratch wound closure. The different treatment groups featured no difference in proliferation and colony forming efficiency (CFE) of limbal epithelial cells or their putative SC marker expression. A significant delay in scratch closure of all the Bevacizumab-treated groups was detected at 4 h. RNA and protein quantification indicated a dose-responsive increase of keratin 3. VEGFA RNA expression also increased while VEGFC and D as well as VEGFR1, 2 and 3 were unchanged. This study highlights previously unknown effects of Bevacizumab on cultured putative limbal epithelial SC: a dose-related increase of keratin 3, an increase in VEGFA as well as a delay in scratch wound closure. These in vitro data should be considered when using Bevacizumab in the context of limbal epithelial SC transplantation.

Local VEGF-A blockade modulates the microenvironment of the corneal graft bed.

The microenvironment plays an important role in several immunological processes. Vascular endothelial growth factor-A (VEGF-A) not only regulates angiogenesis, but is known as a modulator of the immune microenvironment. Modulating the site of transplantation might be beneficial for subsequent transplant survival. In this study, we therefore analyzed the effect that a local blockade of VEGF-A in the inflamed cornea as the graft receiving tissue has on the immune system. We used the murine model of suture-induced neovascularization and subsequent high-risk corneal transplantation, which is an optimal model for local drug application. Mice were treated with VEGFR1/R2 trap prior to transplantation. We analyzed corneal gene expression, as well as protein levels in the cornea and serum on the day of transplantation, 2 and 8 weeks later. Local VEGF depletion prior to transplantation increases the expression of pro-inflammatory as well as immune regulatory cytokines only in the corneal microenvironment, but not in the serum. Furthermore, local VEGFR1/R2 trap treatment significantly inhibits the infiltration of CD11c+ dendritic cells into the cornea. Subsequent increased corneal transplantation success was accompanied by a local upregulation of Foxp3 gene expression. This study demonstrates that locally restricted VEGF depletion increases transplantation success by modulating the receiving corneal microenvironment and inducing tolerogenic mechanisms.

Equine Allogeneic Chondrogenic Induced Mesenchymal Stem Cells Are an Effective Treatment for Degenerative Joint Disease in Horses.

Degenerative joint disease is one of the main causes of equine early retirement from pleasure riding or a performance career. The disease is initially triggered by an abnormal loading of normal cartilage or a normal loading of abnormal cartilage. This primary insult is accompanied with joint inflammation, which leads to further progressive degeneration of the articular cartilage and changes in the surrounding tissues. Therefore, in search for an effective treatment, 75 adult horses with early signs of degenerative fetlock joint disease were enrolled in a randomized, multicenter, double-blinded, and placebo-controlled study. Fifty animals were injected intra-articularly with the investigational veterinary product (IVP) consisting of allogeneic chondrogenic induced mesenchymal stem cells (ciMSCs) with equine allogeneic plasma, and 25 horses were injected with 0.9% NaCl (saline) control product. From week 3 to 18 after treatment, lameness scores (P < 0.001), flexion test responses (P < 0.034), and joint effusion scores (P < 0.001) were remarkably superior in IVP-treated horses. Besides nasal discharge in both treatment groups, no adverse events were observed during the entire study period. On long-term follow-up (1 year), significantly more investigational product-treated horses were working at training level or were returned to their previous level of work (P < 0.001).

Identification of Novel Endogenous Anti(lymph)angiogenic Factors in the Aqueous Humor.

Purpose: The avascular cornea is in direct contact with aqueous humor (AqH). Here we investigate whether AqH exerts anti(lymph)angiogenic effects and thereby may contribute to corneal (lymph)angiogenic privilege. Methods: Using the murine model of suture-induced inflammatory corneal hem- and lymphangiogenesis, the potential anti(lymph)angiogenic effect of AqH was analyzed by applying murine AqH as eyedrops. Anti(lymph)angiogenic effects were measured using morphometric analysis of flat mounts stained with CD31 as panendothelial and LYVE-1 as specific lymphatic endothelial marker. The potential antilymphangiogenic effect of immunomodulatory factors contained in AqH such as vasoactive intestinal peptide (VIP) and α-melanocyte stimulating hormone (α-MSH) was analyzed in lymphatic and blood vascular endothelial cell proliferation assays in vitro. Results: Topically applied AqH significantly inhibited corneal hemangiogenesis and even more so lymphangiogenesis in vivo and directly in vitro. The immunoregulatory factors VIP and α-MSH significantly inhibited lymphatic endothelial cell proliferation in vitro. Depletion of VIP or α-MSH from AqH diminished its anti-hem- and lymphangiogenic potential. Conclusions: Aqueous humor exerts significant antilymphangiogenic effects in vivo. This is at least partially mediated by the known immunomodulatory factors VIP and α-MSH present in the AqH. Therefore, AqH not only contributes to corneal lymphangiogenic privilege and is a new tool to identify novel endogenous regulators of lymphangiogenesis but also may have therapeutic applications.

Short-Term Ultraviolet A Irradiation Leads to Dysfunction of the Limbal Niche Cells and an Antilymphangiogenic and Anti-inflammatory Micromilieu.

PURPOSE: We analyzed the effects of short-term ultraviolet A (UVA) irradiation on the putative limbal stem cell phenotype, limbal fibroblasts, corneal inflammation, and corneal (lymph)angiogenic privilege. METHODS: Primary human limbal epithelial cells and fibroblasts were irradiated with 5.2 J/cm2 of UVA. The limbal epithelial cell phenotype was assessed using P63a, cytokeratin 15, integrin b1 (marking stem and transient amplifying cells), and cytokeratin 3 (a differentiation marker) as well as by a colony-forming efficiency (CFE) assay. An epithelial-fibroblast coculture model was used to compare the ability of irradiated and nonirradiated fibroblasts to support the putative limbal stem cell phenotype. The effects of the conditioned media of irradiated and nonirradiated cells on proliferation and tube formation of human lymphatic and blood endothelial cells also were tested. The levels of factors related to angiogenesis and inflammation were assessed in a protein array and using ELISA. RESULTS: Ultraviolet A induced phenotypical changes of limbal epithelial cells, as their CFE and putative stem cell/transient amplifying marker expression decreased. Limbal epithelial cells cocultured with UVA-irradiated limbal fibroblasts also exhibited differentiation and CFE decrease. Conditioned media from irradiated limbal epithelial cells and fibroblasts inhibited lymphatic endothelial cell proliferation and tube network complexity. Levels of monocyte chemoattractant protein 1 (MCP1) were reduced following UVA irradiation of both cell populations, while levels of IFN-γ increased in irradiated limbal epithelial cells. CONCLUSIONS: These data imply a key role of cellular components of the limbal niche following short-term UVA irradiation. Overall, UVA irradiation leads to dysfunction of these cells and a anti(lymph)angiogenic and anti-inflammatory micromilieu.

Micro-solid-phase extraction coupled to desorption electrospray ionization-high-resolution mass spectrometry for the analysis of explosives in soil.

Home-made micro-solid-phase extraction (SPE) cartridges using different adsorbent materials were tested for the desorption electrospray ionization-high-resolution mass spectrometry (DESI-HRMS) determination of explosives like 2,4,6-trinitrotoluene, cyclotrimethylene-trinitramine, cyclotetramethylene-tetranitramine, pentaerythritol tetranitrate, and trinitrophenylmethylnitramine in soil samples. Quantitation limits in the low nanogram per kilogram range proved the reliability of the method for the detection of explosives at ultra-trace levels. The reduced sample preparation allowed for low costs and high-throughput analyses. Finally, the superior extraction capability of the method was proved by obtaining DESI-HRMS responses at least five times higher than those achieved by performing DESI-HRMS analyses of solid-liquid extracts spotted onto commercial polytetrafluoroethylene slides.

Field clinical study evaluating the efficacy and safety of an oral formulation containing milbemycin oxime/praziquantel (Milbemax®, Novartis Animal Health) in the chemoprevention of the zoonotic canine infection by Dirofilaria repens.

BACKGROUND: Dirofilaria repens is the causative agent of subcutaneous dirofilariosis of dogs, other animals and humans. This nematode is transmitted by mosquitoes of Aedes, Anopheles and Culex genera. In dogs, the parasite may cause subclinical infection or cutaneous signs. Recently, D. repens has emerged and spread in different geographical areas, with an increase of cases in dogs and humans. Chemoprevention in dogs in endemic areas is the most reliable approach for controlling this infection. This paper describes a randomized, blocked and multicentric clinical field study investigating the efficacy of an oral, chewable formulation containing milbemycin oxime/praziquantel (Milbemax®, Novartis Animal Health) in the chemoprevention of subcutaneous dirofilariosis in dogs. METHODS: This study was conducted in endemic areas of Italy. A total of 249 dogs, at two sites, negative for D. repens, were allocated into two groups (i.e. Treated -T1 vs Untreated-T2) with a ratio of 1:1, and subjected to clinical visits and blood sampling once monthly until the end of the study. All blood samples were microscopically and genetically examined. Animals belonging to T1 group received a minimum target dose of 0.5 mg/kg bodyweight of milbemycin oxime and 5 mg/kg of praziquantel in commercial tablets (Milbemax®) according body weight once every 4 weeks. Animals of group T2 were not treated with Milbemax® but received, when necessary, specific parasiticide treatments. The study duration was 336 ± 2 days for each dog. RESULTS: A total of 219 dogs completed the study (i.e. 111 in T1 and 108 in T2), while 30 dogs (i.e. 13 in T1, 17 in T2) were withdrawn for a variety of reasons unrelated to administration of Milbemax®. The percentages of animals not showing microfilariae of D. repens were 100% (111 animals) in T1 and 94.7% (108 animals out of 114) in group T2. Milbemax® was shown to be safe in treated dogs. CONCLUSIONS: The results of this study confirm that the monthly use of Milbemax® in dogs is effective and safe for the prevention of subcutaneous dirofilariosis in endemic areas.

Efficacy and safety of imidacloprid 10%/moxidectin 1% spot-on formulation in the treatment of feline infection by Capillaria aerophila.

The nematode Capillaria aerophila (Trichuroidea, Trichuridae) affects the respiratory system of cats and other animals and occasionally of human beings. Infected cats may show bronchovesicular sounds, inflammation, sneezing, wheezing and, chronic cough and, sometimes, bronchopneumonia and respiratory failure. The present study evaluated the efficacy and safety of the antiparasitic spot-on formulation containing imidacloprid 10%/moxidectin 1 % (Advocate®, Bayer Animal Health) in the treatment of natural feline infection with the lungworm C. aerophila. The efficacy of Advocate® administered once was tested on days 7 ± 1 and 11 ± 1 following treatment at day 0 and compared to faecal egg counts on days -6 ± 1 and -2 ± 1. Overall, 36 cats treated either with Advocate® (treatment group, n = 17 cats) or left untreated (control group, n = 19 cats) were included in the study. Geometric means of faecal egg counts values in eggs per gram of faeces were 124.03 prior to treatment and 0.26 posttreatment in treatment group, while 107.03 and 123.94 pre- and posttreatment in the untreated cats. Post-baseline egg counts showed a 99.79 % reduction in Advocate®-treated animals in comparison with cats which were left untreated. Also, treated cats showed no adverse events. This trial demonstrated that Advocate® spot-on formulation is safe and effective in the treatment of feline lung capillariosis caused by C. aerophila.

MicroRNA 15a, inversely correlated to PKCα, is a potential marker to differentiate between benign and malignant renal tumors in biopsy and urine samples.

NF-κB signal transduction is a potential therapeutic target in many malignant tumors. We have recently shown, in malignant renal proximal tumor cells, that a transcription complex, consisting of NF-κB p65 and mitogen-activated protein kinase p38α, joined by protein kinase C (PKC) α, transmigrates into the nucleus. There, PKCα suppresses the nuclear release of primary microRNA (pri-miRNA) 15a. Induced by endothelin (ET)-1, a decrease in PKCα levels leads to increased miRNA 15a (miR-15A) expression. An identical system can be identified in renal carcinomas, in which, after nuclear transmigration, PKCα binds directly to pri-miRNA 15a in the nucleus. However, the pattern of PKC isoforms differs between malignant renal cell carcinoma (RCC) and benign oncocytoma. PKCα, a component of the transcription complex in tumors, is up-regulated in benign oncocytoma but down-regulated in RCC. Conversely, miRNA 15a is up-regulated in RCC and down-regulated in oncocytoma. A similar behavior is observed in chromophobe carcinoma, whereas differences are less pronounced in papillary RCC (type I): NF-κB target gene expression (ie, ET-1, ET-A and ET-B receptors, vascular cell adhesion molecule-1, IL-6, and fractalkine) is particularly high in malignant RCCs. Up-regulated miRNA 15a can be measured in urine from patients with RCC but is nearly undetectable in oncocytoma, other tumors, and urinary tract inflammation. Thus, the up-regulation of miRNA 15a may be an important marker to help identify malignant clear-cell RCCs in both biopsy and urine samples.

Evaluation of the therapeutic and preventive efficacy of 2.5 % moxidectin / 10 % imidacloprid (Advocate(®), Bayer animal health) in dogs naturally infected or at risk of natural infection by Dirofilaria repens.

The objective of this GCP-compliant clinical field study was to evaluate the efficacy of the combination of moxidectin (minimum dose of 2.5 mg/kg body weight) and imidacloprid (minimum dose of 10.0 mg/kg body weight) spot-on (Advocate(®)) as a preventive and therapeutic treatment of natural infection by Dirofilaria repens in dogs in the Czech Republic.There were two arms of the study, both negatively controlled. 34 animals were randomly allocated to two groups of the treatment arm; 90 negative animals were randomly allocated to the prevention arm groups. All enrolled dogs were observed physically and blood was sampled monthly for Dirofilaria repens microfilaria counts for 18 months by modified Knott test and PCR. 34 dogs were positive for microfilaria and enrolled in the treatment arm of this study (treated: 18, untreated: 16). The reduction of the log-transformed microfilaria counts was significantly higher in the treatment group on day 28 (p = 0.007), 56, 84 and 112 (p < 0.001). All animals treated were negative after a single treatment. In the untreated control group 93.75 % remained positive (p < 0.001). 87 dogs were negative for microfilaria prior to allocation to the "preventive" arm (treated: 49; untreated: 38; 3 excluded). One dog in the untreated control group became positive for Dirofliaria repens microfilaria, while none of the treated dogs became positive. Advocate(®) was effective in the treatment of dogs infected with microfilaria of Dirofilaria repens. Due to the low rate of natural infections the preventive efficacy could not be proven, but no dog treated became positive.

Effectiveness of the homeopathic preparation Zeel compared with carprofen in dogs with osteoarthritis.

The authors compared the symptomatic effectiveness of a complex homeopathic preparation Zeel (1-3 tablets orally per day depending on body weight) to carprofen (4 mg/kg body weight) in dogs (n=68) aged >1 yr diagnosed with osteoarthritis in a multicenter, prospective, observational open-label cohort study in 12 German veterinary clinics. The active treatment period was 56 days. Symptomatic effectiveness, lameness, stiffness of movements, and pain on palpation were evaluated by treating veterinarians and owners. Clinical signs of osteoarthritis improved significantly (P<0.05) at all time points (days 1, 28, and 56) with both therapies. At the end of the treatment period, effectiveness was comparable in both groups. Both treatment regimens were well tolerated with only three treatment-related adverse events, all in the carprofen group.

Effects of commonly used food preservatives on biofilm formation of Streptococcus mutans in vitro.

OBJECTIVE: Sodium benzoate (SB), potassium sorbate (PS) and sodium nitrite (SN) are commonly used food preservatives. In this in vitro study, the effects of these substances on biofilm formation of Streptococcus mutans were analysed. METHODS: In addition to the microtiter plate test (MPT), a biofilm reactor containing bovine enamel slabs (BES) was used to study the influence of food preservatives on biofilm formation in 5 independent periods of 4 days each. These included one period with chlorhexidine digluconate (CHX) as a positive control as well as a period with growth medium alone as a negative control. The vitality of the biofilm on BES was detected using live/dead staining and confocal laser scanning microscopy. Additionally, the number of colony forming units (CFU) was determined. RESULTS: In MPT 0.12% SN significantly reduced the biofilm formation. PS at a concentration of 0.4% tended to inhibit biofilm formation, whereas the inhibition for 0.8% PS was significant. Less inhibition was caused by 0.8% SB. In the biofilm reactor 0.06% of SN, 0.1% of SB and 0.1% PS significantly reduced the covering grade as well as the CFU of the biofilm. Biofilm vitality was reduced significantly by CHX to a level of 32.5% compared to the control. Only SB reduced the vitality to a level of 19.1%. SN and PS showed no influence on biofilm vitality. CONCLUSION: This study indicates the potential of food preservatives as inhibitory agents in S. mutans biofilm formation, which should be kept in mind when studying the effects of conserved food on dental plaque biofilm in situ.

Detection and eradication of microorganisms in root-filled teeth associated with periradicular lesions: an in vivo study.

This study determined the presence of microorganisms by culture and polymerase chain reaction in asymptomatic root-filled teeth with periradicular lesions. Furthermore, a disinfecting regimen using sodium hypochlorite (NaOCl), ethylenediaminetetraacetic acid (EDTA), chlorhexidine digluconate (CHX) irrigation, and calcium hydroxide (Ca(OH)(2)) dressing was assessed. After removal of the root-filling material, specimens of 20 cases undergoing retreatment were sampled. Moreover, the canals were sampled after each step of the disinfecting regimen. Prevalence of microorganisms was 60% by culture and 65% by polymerase chain reaction. In four of those samples (31%), DNA of Enterococcus faecalis was found. After further root canal preparation and irrigation using NaOCl and EDTA, microorganisms could be detected in none of the teeth. Thus, CHX and Ca(OH)(2) could not show further disinfection. In contrast, microorganisms were found in two teeth after the interappointment dressing. It may be concluded that proper root canal preparation and irrigation using NaOCl and EDTA are sufficient for decontamination of the root canal system during endodontic retreatment.