Life cycle assessment of trayless dining

Universities in the United States are applying more sustainable approaches to their dining service operations. "The increase in social consciousness and environmental stewardship on college campuses has spurred an array of new and innovative sustainability programs"(ARAMARK Higher Education 2008). University residence dining is typically cafeteria style, with students using trays to carry food. Studies report that food served without trays substantially reduces food waste and water and electrical consumption associated with washing trays. Commonly, these reported results are estimates and not measurements taken under actual operating conditions. This study utilizes measurements recorded under actual dining service conditions in student residence halls at Michigan Technological University to develop the following: 1) operational-specific data on the issues and potential savings associated with a conversion to trayless dining and 2) life cycle assessment (LCA) cost and environmental impact analyses comparing dining with and without trays. For the LCA, the entire life cycle of the system is considered, from the manufacturing to the usage and disposal phases. The study shows that trayless dining reduces food waste because diners carry less food. The total savings for the diner shifts when not using trays for the standard academic year (205 days), with an average number of 700 diners, is 7,032 pounds of food waste from the pre-rinse area (33% reduction) and 3,157 pounds of food waste from the pan washing area (39% reduction). In addition, for each day of the study, the diners consumed more food during the trayless portion of the experiment. One possible explanation for the increased food consumption during this short duration study could be that the diners found it more convenient to eat the extra food on their plate rather than carrying it back for disposal. The trayless dining experiment shows a reduction in dishwasher water, steam, and electrical consumption for each day of the study. The average reduction of dishwasher water, steam, and electrical consumption over the duration of the study were 10.7%, 9.5%, and 6.4% respectively. Trayless dining implementation would result in a decrease of 4,305 gallons of consumption and wastewater discharge, 2.87 mm BTU of steam consumption, and 158 kWh of electrical consumption for the dinner shift over the academic year. Results of the LCA indicate a total savings of $190.4 when trays are not used during the dinner shift. Trayless dining requires zero CO2 eq and cumulative energy demand in the manufacturing stage, reductions of 1005 kg CO2 eq and 861 MJ eq in the usage phase, and reductions of 6458 kg CO2 eq and 1821 MJ eq in the end of the life cycle.

POST-PROJECT ASSESSMENT OF PIT LATRINES IN RURAL PANAMA

In Panama, one of the Environmental Health (EH) Sector’s primary goals is to improve the health of rural Panamanians by helping them to adopt behaviors and practices that improve access to and use of sanitation systems. In complying with this goal, the EH sector has used participatory development models to improve hygiene and increase access to latrines through volunteer managed latrine construction projects. Unfortunately, there is little understanding of the long term sustainability of these interventions after the volunteers have completed their service. With the Peace Corps adapting their Monitoring, Reporting, and Evaluation procedures, it is appropriate to evaluate the sustainability of sanitation interventions offering recommendations for the adaptions of the EH training program, project management, and evaluation procedures. Recognizing the need for evaluation of past latrine projects, the author performed a post project assessment of 19 pit latrine projects using participatory analysis methodologies. First, the author reviewed volunteers’ perspectives of pit latrine projects in a survey. Then, for comparison, the author performed a survey of latrine projects using a benchmarking scoring system to rate solid waste management, drainage, latrine siting, latrine condition, and hygiene. It was observed that the Sanitation WASH matrix created by the author was an effective tool for evaluating the efficacy of sanitation interventions. Overall more than 75%, of latrines constructed were in use. However, there were some areas where improvements could be made for both latrine construction and health and hygiene. The latrines scored poorly on the indicators related to the privacy structure and seat covers. Interestingly those are the two items least likely to be included in project subsidies. Furthermore, scores for hygiene-related indicators were low; particularly those related to hand washing and cleanliness of the kitchen, indicating potential for improvement in hygiene education. Based on these outcomes, the EH sector should consider including subsidies and standardized designs for privacy structures and seat covers for latrines. In addition, the universal adoption of contracts and/or deposits for project beneficiaries is expected to improve the completion of latrines. In order to address the low scores in the health and hygiene indicators, the EH sector should adapt volunteer training, in addition to standardizing health and hygiene intervention procedures. In doing so, the sector should mimic the Community Health Club model that has shown success in improving health and hygiene indicators, as well as use a training session plan format similar to those in the Water Committee Seminar manual. Finally, the sector should have an experienced volunteer dedicated to program oversight and post-project monitoring and evaluation.

NON-CHROMATOGRAPHIC PURIFICATION OF SYNTHETIC BIO-OLIGOMERS

Synthetic oligonucleotides and peptides have found wide applications in industry and academic research labs. There are ~60 peptide drugs on the market and over 500 under development. The global annual sale of peptide drugs in 2010 was estimated to be $13 billion. There are three oligonucleotide-based drugs on market; among them, the FDA newly approved Kynamro was predicted to have a $100 million annual sale. The annual sale of oligonucleotides to academic labs was estimated to be $700 million. Both bio-oligomers are mostly synthesized on automated synthesizers using solid phase synthesis technology, in which nucleoside or amino acid monomers are added sequentially until the desired full-length sequence is reached. The additions cannot be complete, which generates truncated undesired failure sequences. For almost all applications, these impurities must be removed. The most widely used method is HPLC. However, the method is slow, expensive, labor-intensive, not amendable for automation, difficult to scale up, and unsuitable for high throughput purification. It needs large capital investment, and consumes large volumes of harmful solvents. The purification costs are estimated to be more than 50% of total production costs. Other methods for bio-oligomer purification also have drawbacks, and are less favored than HPLC for most applications. To overcome the problems of known biopolymer purification technologies, we have developed two non-chromatographic purification methods. They are (1) catching failure sequences by polymerization, and (2) catching full-length sequences by polymerization. In the first method, a polymerizable group is attached to the failure sequences of the bio-oligomers during automated synthesis; purification is achieved by simply polymerizing the failure sequences into an insoluble gel and extracting full-length sequences. In the second method, a polymerizable group is attached to the full-length sequences, which are then incorporated into a polymer; impurities are removed by washing, and pure product is cleaved from polymer. These methods do not need chromatography, and all drawbacks of HPLC no longer exist. Using them, purification is achieved by simple manipulations such as shaking and extraction. Therefore, they are suitable for large scale purification of oligonucleotide and peptide drugs, and also ideal for high throughput purification, which currently has a high demand for research projects involving total gene synthesis. The dissertation will present the details about the development of the techniques. Chapter 1 will make an introduction to oligodeoxynucleotides (ODNs), their synthesis and purification. Chapter 2 will describe the detailed studies of using the catching failure sequences by polymerization method to purify ODNs. Chapter 3 will describe the further optimization of the catching failure sequences by polymerization ODN purification technology to the level of practical use. Chapter 4 will present using the catching full-length sequence by polymerization method for ODN purification using acid-cleavable linker. Chapter 5 will make an introduction to peptides, their synthesis and purification. Chapter 6 will describe the studies using the catching full-length sequence by polymerization method for peptide purification.

SYNTHETIC OLIGODEOXYNUCLEOTIDE PURIFICATION VIA CATCHING BY POLYMERIZATION

Large quantities of pure synthetic oligodeoxynucleotides (ODNs) are important for preclinical research, drug development, and biological studies. These ODNs are synthesized on an automated synthesizer. It is inevitable that the crude ODN product contains failure sequences which are not easily removed because they have the same properties as the full length ODNs. Current ODN purification methods such as polyacrylamide gel electrophoresis (PAGE), reversed-phase high performance liquid chromatography (RP HPLC), anion exchange HPLC, and affinity purification can remove those impurities. However, they are not suitable for large scale purification due to the expensive aspects associated with instrumentation, solvent demand, and high labor costs. To solve these problems, two non-chromatographic ODN purification methods have been developed. In the first method, the full-length ODN was tagged with the phosphoramidite containing a methacrylamide group and a cleavable linker while the failure sequences were not. The full-length ODN was incorporated into a polymer through radical acrylamide polymerization whereas failure sequences and other impurities were removed by washing. Pure full-length ODN was obtained by cleaving it from the polymer. In the second method, the failure sequences were capped by a methacrylated phosphoramidite in each synthetic cycle. During purification, the failure sequences were separated from the full-length ODN by radical acrylamide polymerization. The full-length ODN was obtained via water extraction. For both methods, excellent purification yields were achieved and the purity of ODNs was very satisfactory. Thus, this new technology is expected to be beneficial for large scale ODN purification.

The Acropolitan - v. 1, no. 6

In this issue...commencement, Butte, Montana, W. B. Daly, Anaconda Copper Mining Company, Masonic Hall, white-washing the M, Rocky Mountain Garden Club

Endothelial cell protection and complement inhibition in xenotransplantation: a novel in vitro model using whole blood

BACKGROUND: Studying the interactions between xenoreactive antibodies, complement and coagulation factors with the endothelium in hyperacute and acute vascular rejection usually necessitates the use of in vivo models. Conventional in vitro or ex vivo systems require either serum, plasma or anti-coagulated whole blood, making analysis of coagulation-mediated effects difficult. Here a novel in vitro microcarrier-based system for the study of endothelial cell (EC) activation and damage, using non-anticoagulated whole blood is described. Once established, the model was used to study the effect of the characterized complement- and coagulation inhibitor dextran sulfate (DXS, MW 5000) for its EC protective properties in a xenotransplantation setting. METHODS: Porcine aortic endothelial cells (PAEC), grown to confluence on microcarrier beads, were incubated with non-anticoagulated whole human blood until coagulation occurred or for a maximum of 90 min. PAEC-beads were either pre- or co-incubated with DXS. Phosphate buffered saline (PBS) experiments served as controls. Fluid phase and surface activation markers for complement and coagulation were analyzed as well as binding of DXS to PAEC-beads. RESULTS: Co- as well as pre-incubation of DXS, followed by washing of the beads, significantly prolonged time to coagulation from 39 +/- 12 min (PBS control) to 74 +/- 23 and 77 +/- 20 min, respectively (P < 0.005 vs. PBS). DXS treatment attenuated surface deposition of C1q, C4b/c, C3b/c and C5b-9 without affecting IgG or IgM deposition. Endothelial integrity, expressed by positivity for von Willebrand Factor, was maintained longer with DXS treatment. Compared with PBS controls, both pre- and co-incubation with DXS significantly prolonged activated partial thromboplastin time (>300 s, P < 0.05) and reduced production of thrombin-antithrombin complexes and fibrinopeptide A. Whilst DXS co-incubation completely blocked classical pathway complement activity (CH50 test) DXS pre-incubation or PBS control experiments showed no inhibition. DXS bound to PAEC-beads as visualized using fluorescein-labeled DXS. CONCLUSIONS: This novel in vitro microcarrier model can be used to study EC damage and the complex interactions with whole blood as well as screen ''endothelial protective'' substances in a xenotransplantation setting. DXS provides EC protection in this in vitro setting, attenuating damage of ECs as seen in hyperacute xenograft rejection.

Virulence typing of Escherichia coli using microarrays

We describe a microarray based broad-range screening technique for Escherichia coli virulence typing. Gene probes were amplified by PCR from a plasmid bank of characterised E. coli virulence genes and were spotted onto a glass slide to form an array of capture probes. Genomic DNA from E. coli strains which were to be tested for the presence of these virulence gene sequences was labelled with fluorescent cyanine dyes by random amplification and then hybridised against the array of probes. The hybridisation, washing and data analysis conditions were optimised for glass slides, and the applicability of the method for identifying the presence of the virulence genes was determined using reference strains and clinical isolates. It was found to be a sensitive screening method for detecting virulence genes, and a powerful tool for determining the pathotype of E. coli. It will be possible to expand and automate this microarray technique to make it suitable for rapid and reliable diagnostic screening of bacterial isolates.

Assessment of BoviPureTM for the In Vitro Production of Bovine Embryos

The objective of this study was to examine the potential utility of a commercially available sperm separation and purification product for the in vitro production of bovine embryos. Bovine oocytes were purchased from a commercial supplier, and matured oocytes were randomly allocated to one of two treatments. Oocytes were co-incubated with frozen-thawed semen washed twice with BoviPureTM (BoviPure group) or with modified Brackett-Oliphant medium (control group). After a 6-hour insemination period, oocytes were cultured in vitro for 8 days. Cleavage rate of embryos was determined 48 hours post-insemination, and blastocyst formation rate was assessed on day 8 of culture. The experiment was replicated three times, and data were analyzed using chi-square analysis. Washing of sperm in BoviPureTM had no effect (P>.05) on either cleavage rate (77.2%) or blastocyst development (21.6%) when compared with controls (71.9% and 17.1%, respectively). These results indicate that, under conditions of our study, the washing of sperm with BoviPureTM did not significantly enhance the ability to produce bovine embryos in vitro.

A tephrostratigraphic record for the last glacial-interglacial cycle from Lake Ohrid, Albania and Macedonia

ABSTRACT: Here we present a tephrostratigraphic record (core Co1202) recovered from the northeastern part of Lake Ohrid (Republics of Macedonia and Albania) reaching back to Marine Isotope Stage (MIS) 6. Overall ten horizons (OT0702-1 to OT0702-10) containing volcanic tephra have been recognised throughout the 14.94m long sediment succession. Four tephra layers were visible at macroscopic inspection (OT0702-4, OT0702-6, OT0702-8 and OT0702-9), while the remaining six are cryptotephras (OT0702-1, OT0702-2, OT0702-3, OT0702-5, OT0702-7 and OT0702-10) identified from peaks in K, Zr and Sr intensities, magnetic susceptibility measurements, and washing and sieving of the sediments. Glass shards of tephra layers and cryptotephras were analysed with respect to their major element composition, and correlated to explosive eruptions of Italian volcanoes. The stratigraphy and the major element composition of tephra layers and cryptotephras allowed the correlation of OT0702-1 to AD 472 or AD 512 eruptions of Somma-Vesuvius, OT0702-2 to the FL eruption of Mount Etna, OT0702-3 to the Mercato from Somma-Vesuvius, OT0702-4 to SMP1-e/Y-3 eruption from the Campi Flegrei caldera, OT0702-5 to the Codola eruption (Somma-Vesuvius or Campi Flegrei), OT0702-6 to the Campanian Ignimbrite/Y-5 from the Campi Flegrei caldera, OT0702- 7 to the Green Tuff/Y-6 eruption from Pantelleria Island, OT0702-8 to the X-5 eruption probably originating from the Campi Flegrei caldera, OT0702-9 to the X-6 eruption of generic Campanian origin, and OT0702-10 to the P-11 eruption from Pantelleria Island. The fairly well-known ages of these tephra layers and parent eruptions provide new data on the dispersal and deposition of these tephras and, furthermore, allow the establishment of a chronological framework for core Co1202 for a first interpretation of major sedimentological changes.

Bond strength of adhesives to dentin contaminated with smoker's saliva.

The purpose of this study was to determine the effects of contamination with smoker's and non-smoker's saliva on the bond strength of resin composite to superficial dentin using different adhesive systems. The interfacial structure between the resin and dentin was evaluated for each treatment using environmental scanning electron microscopy (ESEM). Freshly extracted human molars were ground with 600-grit SiC paper to expose the superficial dentin. Adhesives [One-Up-Bond-F-Plus (OUFP) and Adper-Prompt-L-Pop (APLP)] and resin composite (TPHSpectrum) were bonded to the dentin (n = 8/group, 180 total specimens) under five surface conditions: control (adhesive applied following manufacturers' instructions); saliva, then 5-s air dry, then adhesive; adhesive, saliva, 5-s air dry; adhesive, saliva, 5-s water rinse, 5-s air dry (ASW group); and adhesive, saliva, 5-s water rinse, 5-s air dry, reapply adhesive (ASWA group). After storage in water at 37 degrees C for 24 h, the specimens were debonded under tension at a speed of 0.5 mm/min. ESEM photomicrographs of the dentin/adhesive interfaces were taken. Mean bond strength ranged from 8.1 to 24.1 MPa. Fisher's protected least significant difference (P = 0.05) intervals for critical adhesive, saliva, and surface condition differences were 1.3, 1.3, and 2.1 MPa, respectively. There were no significant differences in bond strength to dentin between contamination by smoker's and nonsmoker's saliva, but bond strengths were significantly different between adhesive systems, with OUFP twice as strong as APLP under almost all conditions. After adhesive application and contamination with either smoker's or nonsmoker's saliva followed by washing and reapplication of the adhesive (ASWA group), the bond strength of both adhesive systems was the same as that of the control group.

Characterization and mechanism of action of suppressor factors derived from human T cell hybridomas

This laboratory developed human T-cell hybridomas which constitutively secrete suppressor factors (SF) capable of inhibiting immune responses (Hybridoma 6:589 (1987). The mechanisms by which human T-cell hybridoma-derived SFs (designated 160 and 169) and Jurkat leukemic T-cell line derived SF inhibit the proliferative response to mitogen by human PBMC were investigated. The Jurkat SF had a pI of 5.2 whereas the 160 and 169 SF had pI of 5.7 and 4.7 (two peaks) and 4.7, respectively. The SF was not transforming growth factor-beta based upon neutralization and iummunoprecipitation experiments with anti-TGF-beta polyclonal antibody. Il-2 production by human PBMC cultured with Con A or OKT3 mAb in the presence of SF was found to be inhibited by greater than 80%. The proliferative responses of SF treated PBMC could not be restored by addition of exogeneous human IL-2. Inhibition of the proliferative responses could not be reversed by addition of exogenous rIL-1, rIL-2 or rIL-4 alone or in paired combinations. The expression of IL-2 receptors (TAC Ag) on Con A activated cultures time points was not affected by treatment with any SFs. Both the 160 and 169 hybridoma-derived SFs were found to arrest PHA induced cell cycle progression in G$\sb0$/G$\sb1$ phase, whereas SF from the Jurkat T-cell line arrested progression in the S phase. Pretreatment of PBMC with SF prior to the addition of mitogen, followed by washing, did not alter the proliferative response of these PBMC nor their cell cycle progression suggesting that cell activation is necessary for these SF to inhibit proliferative responses. Northern blot analysis of total mRNA from mitogen stimulated PBMC in the presence of SF, revealed a time dependent accumulation of an IL-2 specific mRNA of increased size (2.8 kB) in addition to the expected 1.0 kB mature IL-2 message. Interferon-gamma mRNA was of the appropriate size but its half-life was prolonged in SF treated cultures. IL-2 receptor and IL-1 beta mRNA expression was not altered in these cells. ^

Mycobacterium ulcerans persistence at a village water source of Buruli ulcer patients

Buruli ulcer (BU), a neglected tropical disease of the skin and subcutaneous tissue, is caused by Mycobacterium ulcerans and is the third most common mycobacterial disease after tuberculosis and leprosy. While there is a strong association of the occurrence of the disease with stagnant or slow flowing water bodies, the exact mode of transmission of BU is not clear. M. ulcerans has emerged from the environmental fish pathogen M. marinum by acquisition of a virulence plasmid encoding the enzymes required for the production of the cytotoxic macrolide toxin mycolactone, which is a key factor in the pathogenesis of BU. Comparative genomic studies have further shown extensive pseudogene formation and downsizing of the M. ulcerans genome, indicative for an adaptation to a more stable ecological niche. This has raised the question whether this pathogen is still present in water-associated environmental reservoirs. Here we show persistence of M. ulcerans specific DNA sequences over a period of more than two years at a water contact location of BU patients in an endemic village of Cameroon. At defined positions in a shallow water hole used by the villagers for washing and bathing, detritus remained consistently positive for M. ulcerans DNA. The observed mean real-time PCR Ct difference of 1.45 between the insertion sequences IS2606 and IS2404 indicated that lineage 3 M. ulcerans, which cause human disease, persisted in this environment after successful treatment of all local patients. Underwater decaying organic matter may therefore represent a reservoir of M. ulcerans for direct infection of skin lesions or vector-associated transmission.

Rainfall input, throughfall and stemflow of nutrients in a central African rain forest dominated by ectomycorrhizal trees

Incident rainfall is a major source of nutrient input to a forest ecosystem and the consequent throughfall and stemflow contribute to nutrient cycling. These rain-based fluxes were measured over 12 mo in two forest types in Korup National Park, Cameroon, one with low (LEM) and one with high (HEM) ectomycorrhizal abundances of trees. Throughfall was 96.6 and 92.4% of the incident annual rainfall (5370 mm) in LEM and HEM forests respectively; stemflow was correspondingly 1.5 and 2.2%. Architectural analysis showed that ln(funneling ratio) declined linearly with increasing ln(basal area) of trees. Mean annual inputs of N, P, K, Mg and Ca in incident rainfall were 1.50, 1.07, 7.77, 5.25 and 9.27 kg ha(-1), and total rain-based inputs to the forest floor were 5.0, 3.2, 123.4, 14.4 and 37.7 kg ha-1 respectively. The value for K is high for tropical forests and that for N is low. Nitrogen showed a significantly lower loading of throughfall and stemflow in HEM than in LEM forest, this being associated in the HEM forest with a greater abundance of epiphytic bryophytes which may absorb more N. Incident rainfall provided c. 35% of the gross input of P to the forest floor (i. e., rain-based plus small litter inputs), a surprisingly high contribution given the sandy P-poor soils. At the start of the wet season leaching of K from the canopy was particularly high. Calcium in the rain was also highest at this time, most likely due to washing off of dry-deposited Harmattan dusts. It is proposed that throughfall has an important `priming' function in the rapid decomposition of litter and mineralization of P at the start of the wet season. The contribution of P inputted from the atmosphere appears to be significant when compared to the rates of P mineralization from leaf litter.

Interactions between dodecyl phosphates and hydroxyapatite or tooth enamel: relevance to inhibition of dental erosion.

Tooth surface modification is a potential method of preventing dental erosion, a form of excessive tooth wear facilitated by softening of tooth surfaces through the direct action of acids, mainly of dietary origin. We have previously shown that dodecyl phosphates (DPs) effectively inhibit dissolution of native surfaces of hydroxyapatite (the type mineral for dental enamel) and show good substantivity. However, adsorbed saliva also inhibits dissolution and DPs did not augment this effect, which suggests that DPs and saliva interact at the hydroxyapatite surface. In the present study the adsorption and desorption of potassium and sodium dodecyl phosphates or sodium dodecyl sulphate (SDS) to hydroxyapatite and human tooth enamel powder, both native and pre-treated with saliva, were studied by high performance liquid chromatography-mass Spectrometry. Thermo gravimetric analysis was used to analyse residual saliva and surfactant on the substrates. Both DPs showed a higher affinity than SDS for both hydroxyapatite and enamel, and little DP was desorbed by washing with water. SDS was readily desorbed from hydroxyapatite, suggesting that the phosphate head group is essential for strong binding to this substrate. However, SDS was not desorbed from enamel, so that this substrate has surface properties different from those of hydroxyapatite. The presence of a salivary coating had little or no effect on adsorption of the DPs, but treatment with DPs partly desorbed saliva; this could account for the failure of DPs to increase the dissolution inhibition due to adsorbed saliva.

Antiseptic solutions modulate the paracrine-like activity of bone chips: differential impact of chlorhexidine and sodium hypochlorite

AIM: Chemical decontamination increases the availability of bone grafts; however, it is unclear whether antiseptic processing changes the biological activity of bone. MATERIALS AND METHODS: Bone chips were incubated with 4 different antiseptic solutions including (1) povidone-iodine (0.5%), (2) chlorhexidine diguluconate (0.2%), (3) hydrogen peroxide (1%) and (4) sodium hypochlorite (0.25%). After 10 minutes of incubation, changes in the capacity of the bone-conditioned medium to modulate gene expression of gingival fibroblasts was investigated. RESULTS: Conditioned medium obtained from freshly prepared bone chips increased the expression of TGF-β target genes interleukin 11 (IL11), proteoglycan4 (PRG4), NADPH oxidase 4 (NOX4), and decreased the expression of adrenomedullin (ADM), and pentraxin 3 (PTX3) in gingival fibroblasts. Incubation of bone chips with 0.2% chlorhexidine, followed by vigorously washing resulted in a bone-conditioned medium with even higher expression of IL11, PRG4, and NOX4. These findings were also found with a decrease in cell viability and an activation of apoptosis signaling. Chlorhexidine alone, at low concentrations, increased IL11, PRG4 and NOX4 expression, independent of the TGF-β receptor I kinase activity. In contrast, 0.25% sodium hypochlorite almost entirely abolished the activity of bone-conditioned medium, while the other two antiseptic solutions, 1% hydrogen peroxide and 0.5% povidone-iodine, had relatively no impact, respectively. CONCLUSION: These in vitro findings demonstrate that incubation of bone chips with chlorhexidine differentially affects the activity of the respective bone-conditioned medium compared to the other antiseptic solutions. The data further suggest that the main effects are caused by chlorhexidine remaining in the bone-conditioned medium after repeated washing of the bone chips. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved. KEYWORDS: Autografts; TGF-β; antiseptic solution; bone; bone conditioned medium; bone supernatant; chlorhexidine; hydrogen peroxide; povidone-iodine; sodium hypochlorite