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Journal of Nanomaterials
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Volume 2019, Article ID 5432040, 13 pages
Enhanced Biocompatibility and Antibacterial Activity of Selective Laser Melting Titanium with Zinc-Doped Micro-Nano Topography
Fan Wu,1,2 Ruogu Xu,1,2 Xiaolin Yu,1,2 Jiamin Yang,1,2 Yun Liu,1,2 Jianglin Ouyang,3,4 Chunyu Zhang,3,4 and Feilong Deng1,2
1Department of Oral Implantology, Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-Sen University, Guangzhou, China
2Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China
3Guangzhou Institute of Advanced Technology, Chinese Academy of Science, Guangzhou, China
4Guangzhou Janus Biotechnology Co., Ltd, Chinese Academy of Sciences, Guangzhou, China
Correspondence should be addressed to Feilong Deng; nc.ude.usys.liam@lfgned
Received 28 January 2019; Accepted 2 April 2019; Published 21 May 2019
Academic Editor: Bo Tan
Copyright © 2019 Fan Wu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Selective laser melting (SLM) titanium is a suitable material for use in customized implants. However, long-term implant survival requires both successful osseointegration and promising antibacterial characteristics. Native SLM titanium thus requires proper modifications to improve its bioactivity and antibacterial efficacy. Micro-arc oxidation (MAO) was conducted on sandblasted SLM substrate to form a microporous TiO2 coating. Subsequently, hydrothermal treatment was applied to fabricate micro-nano zinc-incorporated coatings with different Zn content (1 mM-Zn and 100 μM-Zn). Surface characterization was performed using scanning electron microscopy, X-ray photoelectron spectroscopy, X-ray diffraction, a three-dimensional profilometer, and a contact angle measuring device. The osteoblast-like cell line MC3T3-E1, Subclone 14, was used in cell viability assays to evaluate adhesion, proliferation, and ALP activity. An antibacterial assay was conducted using Streptococcus sanguinis and Fusobacterium nucleatum. Zn-incorporated samples exhibited higher adhesion, proliferation, and differentiation than did SLM and MAO samples. 100 μM Zn samples exhibited the highest proliferation, and 1 mM-Zn samples manifested the highest ALP activity. In addition, Zn-incorporated samples exerted inhibitory effects on both Streptococcus sanguinis and Fusobacterium nucleatum. Combining micro-arc oxidation and hydrothermal treatment, we successfully fabricated a novel Zn-incorporated coating on a microporous SLM surface which possesses both outstanding bioactivity and antibacterial efficacy.
Dental implants are the prior choice for patients suffering tooth loss due to predictable clinical outcomes [1]. However, patients whose conditions are complicated by irregular alveolar bone structure or extraction sockets are unsuitable to apply with such implants as their lengths, diameters, and tread parameters are predetermined and unmodifiable [2]. With the rapid development of computer-aided design (CAD) and additive manufacturing (AM), personalized implants can significantly simplify clinical procedure and shorten treatment times. Furthermore, customized implants can minimize stress shielding and pressure-induced bone loss when compared to traditional implants [3, 4]. As manufacturing processes continue to advance, an increasing variety of customized implants are entering clinical use.
Selective laser melting (SLM), one of the most modern types of additive manufacturing (AM), is particularly well suited to fabricate customized implants or bone substitutes with free-form geometry [5–7]. Since the bone-implant interface is crucial to osseointegration, numerous studies have focused on evaluating the biocompatible properties of the SLM surface [8–11]. Compared to conventional machine-processed titanium, the biocompatibility of SLM substrate remains controversial. Shaoki et al. reported that compared to the surface of machine-processed titanium, rates of osteoblast adhesion and differentiation were higher on an SLM surface but residual melted titanium particles decreased removal torque values [8]. Studies have also revealed that bacteria easily adhere to SLM substrate due to its rough and irregular surface [12, 13]. After SLM modifications both in vivo and in vitro (i.e., anodic oxidation, alkali-heat treatment, etc.), remarkable antibacterial properties, bone regeneration, and soft tissue adhesion were demonstrated with an SLM surface [12, 14, 15]. However, few of them possess both biocompatibility and antibacterial activity at the same time.
Micro-arc oxidation (MAO) has been widely used in the modification of biomaterial surfaces and confers a wide variety of biomechanical advantages [16, 17]. MAO-produced porous TiO2 coating, incorporated with calcium and phosphorous, has been found to favorably influence osteoblast adhesion, proliferation, and differentiation [18]. In our previous study, we fabricated a rougher TiO2 coating by combining sandblasting and acid etching technology with MAO, thus significantly enhancing biocompatibility [19]. Layer-by-layer manufacturing of native SLM specimens also resulted in a rough surface. Few studies, however, have focused on modifying the microscale surface of SLM specimens by MAO.
Peri-implantitis caused by bacteria is one severe complication that can eventually lead to implant failure [20]. Bacteria can not only aggregate on the implant surface, forming a biofilm and resisting antibacterial treatment, but also impede osseointegration and form alveolar absorption [21]. In order to enhance the antibacterial properties of biomaterials, various modification strategies have been applied, such as the loading of antibiotics, cross-linking of drugs, and construction of micro-nano topography [12, 22, 23]. Although antibacterial properties have improved, few methods with a focus on improving both osteogenesis and antibacterial properties have been reported.
Zinc, an essential trace element involved in multiple skeletal signaling pathways, reduces osteoclast resorption and improves osteoblast differentiation by regulating the expression of related genes [24, 25]. Biomaterials incorporating Zn have been found to exhibit remarkable osteogenic and antibacterial activity [26–30]. Among the different Zn compounds, ZnO exhibits powerful antibacterial properties due to its membrane-damaging effects, generation of reactive oxygen species (ROS), and release of Zn ions [31]. However, a high concentration of Zn not only is toxic to bacteria but also causes damage to human cells [32]. It is thus vital to appropriately adjust the concentration of Zn in any biomaterial, enough to inhibit bacterial adhesion and reproduction while conferring no cytotoxicity.
In this study, we modified the surface of an SLM substrate using MAO thus forming a microporous morphology. We further doped Zn ions onto the MAO surface by hydrothermal treatment, constructing a micro-nano topography on the SLM specimen. Surface characterization was performed by scanning electron microscopy (SEM), energy-dispersive spectroscopy (EDS), X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), a 3D profilometer, and a contact angle measuring device. Antibacterial activity was assessed by oral pathogenic bacterial species, including S. sanguinis and F. nucleatum. Cytocompatibility of the modified SLM surface was evaluated by characterizing the morphology, attachment, proliferation, and differentiation of osteoblast-like cells (MC3T3-E1). Despite that MAO has been widely used in implant surface modification, here we present a novel method to combine MAO and zinc element of the SLM substrate, which both improves biological performance and enhances antibacterial efficacy with the ultimate aim of improving customized dental implant success rates and reducing the risk of peri-implant inflammation.
2.1. Specimen Preparation and Treatment
The SLM specimens were manufactured as described previously [12, 15]. Briefly, round-disk samples (diameter, 10 mm; thickness, 1 mm) were designed by SolidWorks® 12.0 (SolidWorks Corp, USA). According to the data, specimens were fabricated by an SLM machine (SLM125HL, SLM Solutions GmbH, Germany) using commercial grade II titanium powders (Western BaoDe, China) as raw materials. The main characteristics of SLM machine were as follows: the maximum laser power is 145 W, the layer thickness is 30 μm, the laser scanning speed is 355 mm/s, the laser spot size is 50 μm, and the hatch space is 45 μm. All SLM titanium specimens were ultrasonically cleaned with acetone, ethanol, and deionized water for 15 min each time considered as SLM samples. To eliminate the residual titanium spheres and the native oxide layer of surface, some of the SLM samples were sandblasted by ZrO2 particles (diameter, 250 μm) and then chemically polished in 5% hydrofluoric acid solution for 30 sec. Subsequently, they were ultrasonically cleaned and dried. MAO was conducted in an electrolyte solution with 0.02 M glycerophosphate disodium salt pentahydrate (C3H7Na2O6P·5(H2O) and 0.05 M calcium acetate monohydrate (Ca(CH3COO)2·H2O)) using a pulsed DC power supply (MHWYDM750-20, Nenghua Electrical Co., China). Frequency, duty, voltage, and time were 100 HZ, 26%, 410 V, and 6 min, respectively. These specimens were considered as MAO samples. After MAO, parts of the MAO samples were hydrothermally treated in a Teflon-lined autoclave containing 1 mM and 100 μM of zinc acetate (Zn(CH3COO)2) at 200°C for 3 h. These further treated specimens we termed as 1 mM-Zn and 100 μM-Zn samples. Each sample was ultrasonicated in deionized water after every single treatment step. Samples were divided into four groups, namely SLM, MAO, 1 mM-Zn, and 100 μM-Zn.
2.2. Specimen Characterization
Surface topographies were observed using field-emission scanning electron microscopy (FE-SEM, SU70, Hitachi, Japan). Chemistry compositions of surface were analyzed using an electronic differential system (EDS) equipped with FE-SEM and X-ray photoelectron spectroscopy (XPS, ESCALAB 250Xi, Thermo Scientific, USA). Crystallinity of the coatings was evaluated using an X-ray diffractometer (XRD, PANalytical B.V., X'Pert-PRO, Holland) fitted with a Cu Kα () at 40 kV and 40 mA in the range from 20° to 80° (2θ) with a step size of 0.02. Surface profiles and roughness were scanned with a 3D profilometer (BMT EXPERT, Germany). The static contact angles were determined by an optical contact angle measuring device (OCA40 Micro, Data Physics, Germany). To determine Zn-related characteristics of each sample, 1 mM-Zn and 100 μM-Zn specimens were immersed in 3 ml phosphate-buffered saline (PBS, Gibco, USA) at 37°C for 1, 4, 7, and 14 days successively. At predetermined time points, the collected PBS that contained Zn was analyzed by inductively coupled plasma mass spectrometry (ICP-MS, Agilent 7900, Agilent Technologies Inc., USA).
2.3. Cell Culture
The preosteoblastic cell line MC3T3-E1 subclone 14 (Shanghai Cellular Institute of the Chinese Academy of Sciences, China) was cultured in α-MEM medium (Gibco, USA) containing 10% fetal bovine serum (FBS, Gibco, USA) and 1% antibiotic/antimycotic solution (Gibco, USA) at 37°C under a humidified atmosphere of 5% CO2. The culture medium was changed every 2 days to remove suspended cells.
2.4. Cell Proliferation Activity Assay
Cell counting kit-8 assay (CCK-8, Dojindo, Japan) was used to evaluate the cell proliferation of MC3T3-E1 quantitatively [33]. MC3T3-E1 cells were seeded on sample surfaces at a density of cells ml−1 in 48-well plates and cultured for 1, 4, and 7 days. At these predetermined time points, each sample was rinsed three times with PBS. Adherent cells were incubated with 500 μl 10% CCK-8 mixed with culture medium for 2 h at 37°C. The optical density (OD) was measured at 450 nm on an ELISA reader (Tecan, SUNRISE, Switzerland).
2.5. Alkaline Phosphatase (ALP) Activity Assay
MC3T3-E1 cells were seeded on sample surfaces at a density of cells ml-1 in 48-well plates. After culturing with osteogenic medium (α-MEM medium supplemented with 10 mM β-glycerophosphate, 0.05 mM l-ascorbic acid, and 100 mM dexamethasone) for 14 days, cells were lysed by 0.1% Triton X-100 at 4°C overnight and an ALP assay kit (Jiancheng, China) was subsequently used to evaluate the ALP activity measured at an OD value of 520 nm. A protein assay kit (BCA, Beyotime, China) was used to normalize readings to total protein content measured at a wavelength of 562 nm [33].
2.6. Cell Morphology
MC3T3-E1 cells were seeded on sample surfaces at a density of cells ml-1 in 48-well plates for 2 h and fixed in 3% glutaraldehyde solution at 4°C for 10 h. After dehydration in increasing grades of ethanol (50%, 75%, 90%, 95%, and 100%), drying, and sputter-coating with platinum, samples were observed under FE-SEM.
2.7. Bacterial Culture
Streptococcus sanguinis (ATCC 10556) and Fusobacterium nucleatum (ATCC 10953) were used to evaluate the antibacterial activity. S. sanguinis and F. nucleatum were seeded onto brain heart infusion (BHI) agar and blood agar plates (Huankai Microbial, China), respectively. After incubation under anaerobic conditions at 37°C for 24 and 48 h, a single colony of S. sanguinis was placed into the BHI medium and F. nucleatum was placed into the BHI medium supplemented with hemin (0.5 mg/ml) and menadione (0.1 mg/ml). After incubating under anaerobic conditions for 24 h, bacterial suspensions were diluted to CFU ml-1 using the McFarland scale for bacterial adhesion and antibacterial testing. Each sample was placed in a 24-well plate and immersed in 1 ml diluted bacterial suspension for 24 h under anaerobic conditions.
2.8. Bacterial Morphology
Bacterial morphology on all specimens was evaluated using FE-SEM after incubation for 24 h. Samples were rinsed three times with PBS to remove any suspended bacteria. Each sample was immersed in 3% glutaraldehyde at 4°C for 10 h and then dehydrated in a graded series of ethanol solutions (50%, 75%, 90%, 95%, and 100%) for 10 min. All samples were sputter-coated with platinum.
2.9. Antibacterial Test
To determine the antibacterial characteristics of tested materials, samples were rinsed with PBS after 24 h of incubation and transferred into 3 ml PBS. The density of surviving microorganisms was quantified using an ELISA reader at a wavelength of 600 nm in the collected culture medium. The S. sanguinis and F. nucleatum that adhered to each sample were detached by ultrasonic vibration for 15 min (20 W, 80 kHz) and a subsequent 15 s vortex. After a 3000-fold dilution with PBS, 100 μl of diluted suspension was spread onto BHI agar and blood agar plates, respectively. After incubation in an anaerobic environment at 37°C for 48 h, bacterial colony counts of S. sanguinis and F. nucleatum were obtained. Antibacterial effectiveness of adhered S. sanguinis and F. nucleatum were calculated using the antibacterial ratio % (equation (1)), where represents the bacterial count in contact with SLM samples and represents experiment samples (MAO, 1 mM-Zn, and 100 μM-Zn samples).
2.10. Statistical Analysis
SPSS 24.0 software (SPSS Inc., USA) was used to perform statistical analysis. A four-group comparison was measured via one-way ANOVA followed by a Bonferroni post hoc test for multiple comparison (significance, ). All the experiments were performed in triplicate, and all the data were expressed as deviation (SD).
3.1. Surface Characterization
Surface sample topographies are shown in Figure 1. Some residual unmelted titanium spheres were found on the rough SLM surface while porous structures 2–5 μm in size were found formed on MAO samples. The incorporation of Zn and subsequent hydrothermal treatment did not alter porous surface morphology. On high-power magnification, SLM samples revealed irregular grooves and dots. MAO samples revealed a rather smooth surface while nanoparticles appeared distributed throughout the hydrothermally treated samples. The 1 mM-Zn samples revealed a relatively smaller nanoparticle size (20–30 nm), but their shapes were more uniform than on the surfaces of 100 μM-Zn samples (30–60 nm).
Figure 1: SEM images showing surface topographies of each group (scale bar of (a-d) = 20 μm, scale bar of (e-h) = 100 nm). Low magnifications of SLM, MAO, 1 mM-Zn, and 100 μM-Zn samples (a–d); high magnification of SLM, MAO, 1 mM-Zn, and 100 μM-Zn samples in the red boxes location (e–h).
Sample EDS spectra are shown in Figure 2, and surface elemental compositions are shown in Table 1. Ti and O were observed on all samples. The elements Ca and P were noted on all MAO samples. Compared to MAO samples, Ca and P content was decreased in both 1 mM-Zn and 100 μM-Zn samples.
Figure 2: EDS spectra of the samples. SLM samples only consisted of titanium and oxygen elements (a); MAO samples were composited with titanium, oxygen, calcium, and phosphorus elements (b); titanium, oxygen, different contents of zinc, less calcium, and phosphorus elements existed on the surface of 1 mM-Zn and 100 μM-Zn samples (c, d).
Table 1: EDS analysis of the elemental composition of each sample (wt. %).
XRD patterns (Figure 3) confirmed that single-phase anatase TiO2 formed during MAO and MAO with hydrothermal treatment in Zn(CH3COO)2 solution. No Zn- or CaP-containing species were detected by XRD analysis, likely due to their low content or amorphous form in these as-prepared samples.
Figure 3: XRD spectra of each sample. Titanium and anatase were detected on SLM, MAO, 1 mM-Zn, and 100 μM-Zn samples.
XPS analysis (Figure 4) was performed to evaluate the surface chemical states of as-prepared samples. In MAO samples, two peaks (at 532.5 and 530.9 eV) were observed in O 1s XPS spectrum, assigned to the binding energy of O in hydroxyl and lattice oxygen. Peaks at 458.6 eV and 464.3 eV in the Ti 2p XPS spectrum were, respectively, assigned to Ti4+ 2p3/2 and Ti4+ 2p1/2. These XPS findings were in agreement with XRD analysis, confirming that the TiO2 formed during the synthetic procedure. Peaks at 347.6 and 351.2 eV in Ca 2p XPS spectrum, respectively, corresponded to Ca2+ 2p3/2 and Ca2+ 2p1/2. The binding energy at 133.8 eV for P 2p was assigned to the p5+ in Ca10(PO4)6(OH)2 [34]. Compared to MAO samples, no obvious changes in the binding energy of P 2p and Ca 2p were observed in the 100 μM sample. The Zn 2p3/2 at 1021.5 eV and 2p1/2 at 1044.5 eV suggested that Zn2+ content was successfully coated on the surface of TiO2. The O 1s could additionally be split into three peaks: 532.5 eV for hydroxyl in Ca10(PO4)6(OH)2, 529.9 eV for lattice oxygen in TiO2, and 530.9 eV for lattice oxygen in ZnO. The greater binding energy of Zn-O than that of Ti-O is attributed to the greater electronegativity of Zn (1.65) than Ti (1.54). Our findings imply that the layer coating TiO2 was composed of ZnO and Ca10(PO4)6(OH)2.
Figure 4: XPS spectra of MAO and 100 μM-Zn samples. Ti 2p (a), O 1s (b), Ca 2p (c), and P 2p (d) of MAO sample; O 1s (e) and Zn 2p (f) of the 100 μM-Zn sample.
Surface profile images and sample hydrophilicity as determined by water contact angle analysis are shown in Figure 5; findings are shown in Table 2. SLM samples exhibited the highest Ra value of 6.96 μm. The Ra value of MAO, 1 mM-Zn, and 100 μM-Zn samples were 1.85, 1.74, and 1.85 μm, respectively, and were smoother than SLM samples. The water contact angles of SLM and MAO samples were 59.07° and 15.7°, respectively, while 1 mM-Zn and 100 μM-Zn samples exhibited total hydrophilicity and their water contact angles could not be measured.
Figure 5: Surface profiles and contact angles of each sample. Surface roughness profiles of SLM, MAO, 1 mM-Zn, and 100 μM-Zn samples (a–d); MAO samples (f) showed better hydrophilia than did SLM samples (e); 1 mM-Zn and 100 μM-Zn samples (g and h) showed total hydrophilicity.
Table 2: Surface roughness and water contact angles of each sample.
The Zn ion concentration released in PBS was measured by ICP-MS (Figure 6). During immersion in PBS for 14 days, both Zn-incorporated samples released Zn ions continuously at a low level. The total amount of Zn ions in 1 mM-Zn samples was higher than in 100 μM-Zn samples which were in accordance with the surface EDS analysis above.
Figure 6: Zn ion concentration released from each sample of different immersion time.
3.2. Cell Proliferation and Morphology
Figure 7 shows results of MC3T3-E1 proliferation on each sample. At days 1, 4, and 7, MAO, 1 mM-Zn, and 100 μM-Zn samples were found to have greater proliferation than were SLM samples. The 100 μM-Zn group showed higher proliferation than did other groups on the 1st and 7th days. MC3T3-E1 cells exhibited a spindle and elongated shape on SLM surfaces but cellular morphology on MAO, 1 mM-Zn, and 100 μM-Zn samples extensively varied (Figure 8).
Figure 7: Cell proliferation of MC3T3-E1 cells on the surface of each sample after 1, 4, 7 days. and compared with SLM; and compared with MAO; and compared with 1 mM-Zn.
Figure 8: SEM morphology of MC3T3-E1 cells cultured on each sample after 2 hours (scale bar of (a–d) = 2 μm). Red arrows point out the position of cells. SLM (a), MAO (b), 1 mM-Zn (c), and 100 μM-Zn (d).
3.3. Alkaline Phosphatase (ALP) Activity
The osteogenic function of MC3T3-E1 cells was evaluated by analyzing ALP activity after 14 days of culture (Figure 9). SLM samples performed poorly compared to the other three groups in the ALP activity test. ALP activities in 1 mM-Zn and 100 μM-Zn groups were statistically significantly higher than in the MAO group, and the 1 mM-Zn group was found to have the highest statistically significant level (1 mM-Zn > 100 μM-Zn > MAO > SLM).
Figure 9: ALP activity of MC3T3-e1 cells on each sample after 14 days of incubation. and compared with SLM; and compared with MAO; and compared with 1 mM-Zn.
3.4. Antibacterial Activity
Antibacterial findings are detailed in Figure 10. Both S. sanguinis and F. nucleatum adhered to SLM samples at rates significantly higher than in other groups. The antibacterial efficacy of MAO samples against adhered F. nucleatum was ; however, no antibacterial effects of MAO samples against S. sanguinis were found despite SLM samples possessing the roughest surface. Samples incorporated with Zn significantly enhanced antibacterial activity with 1 mM-Zn group samples exhibiting the most effective inhibition of both bacterial species. Morphologies of S. sanguinis and F. nucleatum captured by SEM (Figure 11) revealed how samples incorporated with Zn also reduced bacterial adhesion. Lower quantities of S. sanguinis and F. nucleatum were found on 1 mM-Zn and 100 μM-Zn sample surfaces, while organisms found adhered to SLM and MAO samples were densely aggregated.
Figure 10: Antibacterial ratio of S. sanguinis and F. nucleatum on the surfaces of each sample after 24 h of incubation. and compared with the baseline (0% as SLM group). and compared with MAO.
Figure 11: SEM morphology of S. sanguinis and F. nucleatum adhesion on the surface of each sample after 24 hours of incubation (scale bar of (a–h) = 2 μm). SEM of S. sanguinis: SLM (a), MAO (b), 1 mM-Zn (c), and 100 μM-Zn (d), and SEM of F. nucleatum: SLM (e), MAO (f), 1 mM-Zn (g), and 100 μM-Zn (h).
Bacterial density in Zn-incorporated sample culture media was statistically significantly lower than in SLM and MAO groups (Figure 12). Compared with 100 μM-Zn samples, bacterial density in 1 mM-Zn samples was markedly lower, implying that bacterial density gradually decreased as the concentration of Zn ions in supernatant increased.
Figure 12: The bacteria density in supernatant of S. sanguinis and F. nucleatum after 24 hours of incubation. S. sanguinis (a) and F. nucleatum (b). and compared with SLM; and compared with MAO; and and compared with 1 mM-Zn.
Surface modification of titanium can significantly enhance the osteogenesis and antibacterial efficacy [35–38]. In this study, we fabricated a novel Zn-incorporated TiO2 coating by MAO and subsequent hydrothermal treatment producing micro-nano topography on an SLM surface.
Surface roughness plays an important role in the adherence and proliferation of cells and bacteria. Albrektsson and Wennerberg demonstrated that dental implants with moderately rough surfaces () evoke stronger bone responses than do smoother or rougher surfaces [39]. Rønold and Ellingsen [40] and Rønold et al. [41] proved that bone bonding forces increase with rougher surfaces to a threshold of 3.62–3.90 μm (range: 0.63 μm–11.03 μm). Increased roughness also promotes the formation and maturation of biofilms, especially when the Ra value exceeds 0.2 μm [42, 43]. Due to the layer-by-layer manufacturing process and unmelted spheres, the original SLM specimens we fabricated had an Ra value of 6.96 μm. In order to decrease substrate roughness, we applied sandblasting and acid polishing to eliminate residual particles prior to the MAO procedure and subsequent hydrothermal treatment. The Ra values of MAO, 1 mM-Zn, and 100 μM-Zn samples were 1.85 μm, 1.74 μm, and 1.85 μm, respectively, all within the range of optimal implant surface roughness.
Surface wettability is another important physicochemical property of biomaterials. Previous studies have shown that the hydrophilicity of surfaces influences both cellular and bacterial morphology, adherence, and proliferation. Compared to hydrophobic surfaces, hydrophilic surfaces significantly promote cellular attachment and proliferation [44]. In addition, implants with significant wettability decrease the postimplant healing period and accelerate early osseointegration [45–47]. MAO, 1 mM-Zn, and 100 μM-Zn samples were all evaluated with high surface hydrophilicity by water contact angle analysis. Most oral bacteria, including S. sanguinis and F. nucleatum, thrive better on surfaces with high wettability [48, 49]. Further modifications should be applied to improve antibacterial effects.
Zn-incorporated coatings are well known to possess strong bacteriostatic properties, effectively inhibiting adhesion and proliferation of a wide variety of bacteria [26–28]. The ZnO in coatings also exerts antibacterial effects by ROS generation (i.e., H2O2, OH-, and O22-) [50]. In addition, free Zn ions also penetrate bacterial membranes and cause DNA damage [51]. Research has proven that dental implant failure was commonly associated to dental plaque formation. As an early colonizer of dental plaques, S. sanguinis can attach directly to the surface of implants and facilitate the adhesion of later colonized bacteria, which eventually transfer the plaque to a "mature" community with high levels of Gram-negative anaerobic filamentous organisms [52, 53]. F. nucleatum, one of the most common pathogens associated with peri-implantitis, is a later colonizer during biofilm formation [54]. We also noted that the antibacterial efficacy towards both S. sanguinis and F. nucleatum depended on the content of Zn within coatings. This is likely due to coatings with a higher Zn content releasing a greater quantity of Zn ions and more strongly inhibiting bacterial adhesion and proliferation by mechanisms such as ROS formation. In contrast to S. sanguinis, colony counts of F. nucleatum adhered to MAO samples were significantly lower than those of SLM samples. This difference is likely due to the catalytic effect of anatase in MAO coatings [55].
As a trace element, Zn plays a crucial role in the process of bone regeneration. Various studies have reported that biomaterials incorporating Zn exhibited promising biological characteristics both in vivo and in vitro [29, 30]. Zn enhances osteoblastic adhesion and proliferation by upregulating the expression of FAK and integrin [56]. Zn exerts positive effects on bone formation by promoting the expression of genes involved in osteogenesis such as Runx2, ALP, OCN, and Col-I [30]. Moreover, Zn also decreases bone loss by suppressing osteoclast differentiation via inhibition of RANK expression [57]. Importantly, Zn can only confer favorable cytocompatibility properties and minimal cytotoxicity at an extremely low concentration. Yamamoto et al. demonstrated that the half maximal inhibitory concentration (IC)50 of ZnCl2 is /l (equals to 12.65 ppm of Zn ions) [58]. Ito et al. additionally reported that no cytotoxicity was observed at a Zn ion concentration of 3 ppm [59]. As shown in Figure 6, Zn ions released from 1 mM-Zn and 100 μM-Zn samples accumulated to levels lower than 3 ppm and thus remained in a relatively safe range.
In our present experiment, MC3T3-E1 cells were used to test Zn-incorporated coating viability. SEM images revealed that both 1 mM-Zn and 100 μM-Zn samples were favorable for cell adhesion. Cells were observed extensively spreading out on Zn-incorporated coating surfaces. In addition, first-day CCK-8 results revealed that cells proliferate better on Zn-incorporated samples when compared to SLM and MAO samples, likely due to the outstanding hydrophilicity and special micro-nano topography of the former groups. After incubation for 7 days, 100 μM-Zn samples were found to be the most ideal for cell proliferation. MAO and 1 mM-Zn samples exhibited similar biological characteristics and were both superior to SLM specimens.
ALP activity, an early marker of osteogenesis, was markedly increased on Zn-incorporated specimens compared to SLM and MAO samples. Moreover, 1 mM-Zn samples expressed the highest ALP activity after 14 days, suggesting that higher loading of incorporated Zn exerts positive effects on cellular differentiation as long as Zn content does not exceed a concentration that is biologically safe. These results were not completely in accordance with the cell proliferation assays in which 100 μM-Zn samples showed the best proliferation ability. This might be because a high content of ZnO from Zn-incorporated samples could not only enhance ALP activity but also decrease the proliferation of cells in some degree at the same time despite the suitable surface topography, roughness, and hydrophilicity, which drew no conflicting conclusions to these two results.
With MAO and subsequent hydrothermal treatment, varying quantities of Zn can be incorporated into TiO2 coatings to modify the native SLM surface. Compared with SLM and MAO specimens, Zn-incorporated samples exhibited better antibacterial activity and cytocompatibility.
Further studies should focus on animal experiments to evaluate whether this Zn-incorporated modification possesses similar biological capabilities in vivo as it does in vitro, thus establishing a solid foundation for future clinical application.
In this study, a novel micro-nano topographic coating incorporated with Zn was fabricated on the rough SLM surface by MAO and hydrothermal treatment. Due to a sustained release of Zn ions and superior hydrophilicity, Zn-incorporated samples highly promoted the adhesion, proliferation, and differentiation of MC3T3-E1 cells when compared to SLM and MAO samples. This modified surface also exhibited outstanding antibacterial effects against oral pathogenic flora (S. sanguinis and F. nucleatum), potentially significantly reducing the risk of peri-implant inflammation and ensuring long-term implant survival. Our study thus contributes to the advancement of customized biomedical implant fabrication with SLM technology.
All data included in this study are available upon request by contact with the corresponding author.
There is no conflict of interest regarding the publication of this paper.
Fan Wu and Ruogu Xu contributed equally to this work.
The authors are sincerely grateful to all the research staff members at the Department of Oral Implantology, Guanghua School of Stomatology, and the Institute of Advanced Technology, Chinese Academy of Science. This work is supported by the National Natural Science Foundation of China (81501600) and Science and Technology Major Project of Guangdong Province (2017B090912004).
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Posted August 12, 2015 by GAMERamble in PC Game Reviews, PC Reviews
Gameplay 6
Submerged provides a relaxing and compelling experience without requiring quick reflexes or lateral thinking. Some players will find this boring and repetitive, while others will revel in the tranquil atmosphere and beautiful environments. Despite the climbing and exploration Submerged is closer to titles such as Dear Esther instead of Assassins Creed, but it is definitely something fans of the genre will enjoy.
Gameplay: Submerged offers a stress-free and tranquil experience that is devoid of combat or puzzles.
Graphics: The water and lighting effects are great, and the environments look downright beautiful at times.
Sound: The haunting soundtrack is a great match for the atmosphere of the game
Summary 7.3 Great
Summary rating from user's marks. You can set own marks for this article - just click on stars above and press "Accept".
Summary 0.0 Terrible
Developer: Uppercut Games Pty Ltd | Publisher: Uppercut Games Pty Ltd | Release Date: 2014 | Genre: Action / Adventure / Indie | Website: Official Website | Purchase: Steam
Submerged is the tale of Miku, a young girl struggling to tend to her wounded brother in a world that is all but submerged in water. The game opens with the pair arriving at a mysterious city with their small fishing boat and from there Miku sets out on a search for the supplies needed by her sibling. It is a game without enemies or combat and also without the risk of death or failure. Instead, you are free to explore the flooded streets by boat before clambering on to the half-submerged buildings and locating the needed supplies.
Submerged is viewed in third person and all the environments are set outdoors which is a nice change of pace. Instead of exploring the usual dark tunnels or caves favored by the genre the focus is purely on navigating your boat to points of interest and then scaling the sides of the towering buildings. A handy telescope allows you to spot far-off supplies, secrets or boat parts and these are then marked on your map. While this gives you a lot of freedom to simply explore and soak up the atmosphere, it also means that there isn't a lot of variety in your objectives. Simply collecting all the required supplies makes for a very short experience, which is why we recommend seeking out all the secrets. The "secrets" are simply basic sketches that provide an overview of what happened to the gameworld, but piecing together the full story make it worth finding them all.
Despite the poor health of her brother, Miku doesn't have any time limits to contend with, so you are free to explore at your own leisurely pace. The day and night cycle, as well as weather effects actually means it is very relaxing to just sail around aimlessly and check out the environments. Although there aren't any enemies you'll still encounter some wildlife, such as whales, dolphins, manta rays, birds and other aquatic creatures. Sailing around with a school of dolphins flanking your little boat and a giant whale breaching the water right in front of you is quite an experience, but unfortunately there are only a total of 8 different species in the game.
Submerged runs on the Unreal 4 engine and at times look downright beautiful. During certain times of the day, such as sunrise or sunset, the environments look absolutely magical, but can also look a little drab under certain light conditions. The water and lighting effects are top notch though, and there is even a "post-card" feature that allows you to capture those picture perfect moments while you explore.
Speaking of exploration, Submerged only features about ten large buildings that need to be scaled in order to collect the supplies and a couple of smaller ones where secrets are hidden. Climbing the ledges, pipes, ladders and vines that provide footholds is as easy as holding down the correct directional buttons. No extra buttons are required to jump or anything like that which makes things much easier than in other games like the Assassins Creed series. You cannot fall or die in the game either, which removes all of the tension that usually comes with scaling very high structures. Some players will love this as it keeps the game very relaxing while others will miss the challenge. All the paths and footholds to reach the supplies on top of the buildings are also marked quite clearly, so getting lost or stuck is not really an option.
One of our favorite parts of the game is the soundtrack, which features some beautiful tunes. Fans of piano driven tracks will love the musical score of Jeff Van Dyck as it adds to the tranquil atmosphere of the game. The game also features plenty of sound effects, such as the noises made by the sea creatures or the wind howling around you as you climb the higher buildings. The straightforward controls ensure that the game is just as playable using a controller as it is with a mouse and keyboard setup.
We really enjoyed our time with Submerged, but there is no doubt that players looking for a challenging title with varied objectives will be disappointed. Submerged is not the type of game that requires quick reflexes or puzzle solving skills to complete, which means those not in search of a relaxing experience could find it too repetitive. It is definitely a game where you want to take your time and explore everything as simply collecting all the supplies will leave you staring at the credits in no time. Anyone who enjoys slower paced and relaxing games, such as Dear Esther and Home is Where One Starts should definitely check out Submerged though.
Minimum PC System Requirements
Recommended PC System Requirements
OS: Windows 7 64Bit
Processor: Dual Core 2.0+ GHz
Graphics: GeForce 9800GTX or ATI Radeon HD 4770
Hard Drive: 3 GB available space
Processor: Intel i5 2.5 GHz
Graphics: GeForce GTX 760 or Equivalent card
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Tags: Adventure, Indie, PC, Review
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Deathsmiles
Combine the gothic art style of the early Castlevania games and mix it with the frantic bullet hell gameplay CAVE is so good at and you get Deathsmiles. Although it has been many years since the game first graced arcades and the Xbox 360 its arrival on the PC is still a cause for celebration. The loss of online coop and lack of PC extras is a pity, but the game will still put a smile on any shooter fan's face. Gameplay: Not the hardest game in the genre, but attaining a highscore still takes skill. Graphics: The arcade mode visuals look a bit dusty, but the rest of the game still look good considering its age. Sound: The soundtrack is nice and energetic while the sound effects are good without being overwhelming.
Reverse Crawl
Nerdook once again impresses with a title that delivers turn-based strategy in an engaging, humorous and very accessible manner. The visuals are charming, the gameplay addictive and although short, there are enough reasons to replay the game a couple of times. Anyone looking for a turn-based strategy title that can be enjoyed without a degree in military tactics should definitely add Reverse Crawl to their library. Gameplay: Easy to play, but with enough depth to keep it interesting right to the end. Graphics: Features the typical charming Nerdook visual style. Sound: No voice acting, but the music is pleasant enough.
Knightmare Tower
Despite the fact that you can play the Flash version of the game for free, this version is still worth every penny of the very reasonable asking price. The gameplay is polished to a fine sheen and will keep you busy until you have conquered the tower and unlocked every single item. The only problem is that afterwards you will be left wanting more. Gameplay: Very accessible and very, very addictive. Graphics: Colorful and humorous. Sound: An excellent soundtrack by Hyperduck Soundworks.
Running around as a firefighter extinguishing flames and rescuing civilians is a lot more fun than it sounds and Flame Over packs quite a challenge to boot. It is definitely a little frustrating at times, but the randomized levels and addictive gameplay will keep you coming back for more after every rage quit. The game made the transition from PS Vita to PC very well and comes highly recommended. Gameplay: Fun, sometimes frustrating, very challenging and definitely addictive. Graphics: Four different environments and some very nice fire effects. Sound: Very catchy and fits the theme of the game perfectly.
It will take you a while to conquer the 40 levels of platform puzzles in A-Men but it is worth it if you are up for a challenge. The gameplay feels like something out of the 16bit era, but with a nice coat of modern paint. While some players might relish the challenge it is also bound to frustrate those blessed with less patience. Gameplay: If you are in the mood for a challenging puzzle platformer this is not a bad choice. Graphics: Old school 2D visuals with a unique style. Sound: The music isn't too bad but the voice acting can be a bit hit or miss.
Even if you don't know your shoggoths from your Mi-go there is a lot to like about this Lovecraftian horror title by Zoetrope Interactive. They are no strangers to the genre and are clearly huge fans of H. P. Lovecraft, which is very evident in this game. The slower place and lack of combat might scare away some horror fans, but the attention to detail and the way that the game perfectly captures the atmosphere of Lovecraft's work makes it a must play for fans. It might lack a little polish in some areas and it is over all too soon, but it is still a remarkable effort by a small indie team and one that deserves to be played. Gameplay: Plenty of exploration and puzzle solving in environments that perfectly capture the style of H. P Lovecraft. Graphics: Your surroundings are detailed and quite atmospheric. Sound: The voice acting could have been better, but the rest of the audio is very good.
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redpajama_set_name: RedPajamaCommonCrawl


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Man gets 10-year imprisonment for sexually assaulting girl
28 December 2020, 10:55 am ·1-min read
Thane, Dec 28 (PTI) A special court in Thane district of Maharashtra last week sentenced a 27-year-old man to rigorous imprisonment for ten years for sexually assaulting a four-year-old daughter of his neighbour in Kalyan city, as per a detailed order made available on Monday.
Protection of Children from Sexual Offences (POCSO) Act court judge SR Pahade also imposed a fine of Rs 15,000 on the convict, Gitesh alias Pintya Baban Bansode, after finding him guilty under section 376(2)(i) of the Indian Penal Code (IPC) and sections 4 and 6 of the POCSO Act, 2012.
The court also directed the Maharashtra government to pay adequate compensation to the victim girl under section 357 A of the CrPC.
Bansode had sexually abused the girl at his house in Kalyan on January 26, 2018 after luring her with snacks. He also threatened her against disclosing the incident, the prosecution told the court.
'Sexual violence, apart from being a dehumanising act, is an unlawful intrusion of the right to privacy and sanctity of a female. It is a serious blow to her supreme honour and offends her self esteem and dignity; it degrades and humiliates the victim and where the victim is a helpless innocent child, it leaves behind a traumatic experience,' the court said in its order. PTI CORR NSK NSK


Those who follow the biotech world may have seen that Regeneron reported some fantastic results on its lipid-lowering drug. Much more efficacy than statins. Safety data was not reported but it seems safe to say that suggestions of hepatotoxicity that first appeared with the early statins and seem to be a fixture in use of statins for the first 6 months is a segment of the population, were not present; FDA wouldn't have hesitated to intervene if there had been such suggestion, since in FDA's eyes there are already "safe" lipid-lowering drugs on the market.
The same is true for rhabdomyolysis–essentially break down of muscle (some thing the muscle soreness often associated with statin use may be a pre-rhabdomyolysis state, but the data are anything but clear on that). It was rhabdomyolysis that was the reason Bayer's Baychol was withdrawn from the market.
1. There has long been the observation that if cholesterol levels are brought below 90-100, there is little gain in mortality (some studies suggest there may even be an increase) and that cancer risk in particular goes up. Of course, recovering from a heart attack has a higher probability than doing so from cancer. Unfortunately, I can't tell you which sites–I just don't remember.
2. There are some established drugs in the lipid-lowering space. Lipitor and Crestor come to mind. The former is generic, and it is probably finally making it onto many P&T committee lists. The latter is still patent protected and, not surprisingly, costs a bit more than the former. That's not to say that Crestor is more efficacious than Lipitor. In a given patient, one may prove to be less efficacious or better tolerated than the other. YMMV.
3. The thing that made the statin market what it is today was a series of studies by Bristol-Myers Squibb and Merck showing that use of statins was associated with lower mortality–quite a bit in fact. Since the effect of Pravachol from BMS in mortality reduction was greater than might be expected if only lipid-lowering were the explanation, there has been a persistent question over what exactly it is that statins are doing besides lowering lipids. There are suggestions that they reduce chronic inflammation (considered part of the pathophysiological process underlying atherosclerosis), reduce risk of osteoporosis (very controversial), reduce risk of gingivitis and periodontitis (Dr. Zussman is better positioned to opine on that one than I am), and some suggestions of reduced risk of Alzheimer's disease, among others. Will Regeneron's drug do any of these? We don't know. Will it even lower mortality? Again, we don't know. Such studies take some time to complete, and I'm not sure if they've even been started. There's also the comparative effectiveness matter. How much this drug will cost for each QALY (quality-adjusted life years) gained isn't yet know, and whether the drug is seen to be as good a value as the statins were when they first came to market isn't known. However, make no mistake, all of these factors will enter into the calculus of how successful, if at all, Regeneron's drug might be.
4. As one who has taken Lipitor for 13 years (horrible family history of heart disease–I keep my total cholesterol below a 100 and LDL below 75), I'm not particularly interested in switching drugs, never mind drug classes. There are many patients taking statins who, I think it's probable, think likewise. That most statins are now well off patent (and cheap generics) is another reason to stay with something of known efficacy in a particular patient. That presents a problem for Regeneron: How to convince physicians to put new patients on its drug and to convert those on statins to switch. The former may be straightforward, though the issue will be one of how much more growth can there/will there be in the lipid-lowering market. I'm agnostic-to-skeptical that there's a whole group of patients needing another lipid-lowering drug. That's not to say there aren't some, though. On the other hand, obtaining health insurance coverage may be problematic, as I'm sure that Regeneron will price the drug in the "near and dear" category (to use industry parlance), meaning high. Very high. I doubt that Regeneron will follow the Pfizer strategy of pricing the drug 10% below the leading statins (or in this case, perhaps, Crestor) for two years to gain some traction in the market, but I could be wrong. One thing to consider is that biotechs are not used to pricing competitively. Usually, they are the long entity in a market space, and they will price accordingly. As for switching patients off of statins, I guess if there are those not getting enough of a reduction, perhaps with LDLs over 140-150, there's a chance of a switch being made. There aren't that many of such people, though. All of this means that Regeneron will have some work cut out on the marketing end to get newly diagnosed hyperlipidemics onto its drug, as well as getting insurance reimbursement.
The long and short of it is the Regeneron's drug may be a game-changer in heart disease–but we just don't know enough as yet about it. The data released yesterday seem compelling, yet they are only in terms of reduction in lipid levels. Fine, except we know from the statins that something may be needed to get much benefit from a lipid-lowering drug.
For those of you liking growth stocks or story stocks, this is a company with a nice story to follow, perhaps to take a position in. For value investors (read: Mr. Melvin), enjoy using the drug (if it gets to market) but don't even thing about looking at this stock. It won't be a "value" one for a decade or two at least.
I wonder if any studies have been done on the increased cancer risk. A little while ago, a scientist did a study and claimed that a cancer cure could save something like $5 trillion a year. However, tagged on to the end of the study was a one sentence disclaimer to the effect that the suggested savings did not take into account that while a cancer cure could well cut down on costs, survivors might find that their longer life brought on equally (or more) expensive disabilities - like diabetes or, more likely various dementias. I've been in two post-operative cardiac exercise programs - both for several years. In that time, quite a few individuals come through - most stay for the minimum period; others, like myself continue on. One thing we long-timers watched for was the continued health of those who stayed and, if possible, those who left. The nurses at one hospital were especially diligent in keeping in touch with members of both groups. Over the years (18 to be exact), as one would expect, there have been numerous deaths. However, very, very few were due to cardiac problems - more often, cancer was the cause. So, here's the question: is the propensity for cancer among cardiac survivors an inevitable result of their survival, or can (and should) their deaths be attributed to statins. I know the latter is a popular one, but hell's bells, lawyers couldn't make a dime if it proved out that longevity was the real cause.
Life should be more expensive than death because it is more valuable, especially to survivors. The problem is that the disease lottery is zero sum: you will die of something. As medical / nutrition science advances, death rate due to some diseases has plummeted - and survivors go on to die of something else.
The progress made with infectious and cardiovascular disease has been faster than cancer treatment (and cheaper). So don't smoke, eat fish, hit the gym, wash your hands, and prepare for the final fight with unregulated cell division.
Henri Cartier-Bresson, arguably the most powerful street photographer in history, used only a Leica 3 with a 50mm f3.5. I enjoy his photo book very much.
Robert Doisneau is great too.
Eugene Atget photographed Paris years before Cartier-Bresson was even born. Both had a keen eye, and their photographs of Paris embody a quiet beauty that remains timeless.


Another game which has earned the Steam Greenlight seal of approval is the ZX Spectrum inspired game ' Deep Ones ', which is coming to a PC near you this Summer 2017. In this game you play as a diver who has suffered a terrible crash in his Submarine caused by one big menacing red octopus. You must traverse dangerous underwater caverns, rip and tear through thick plants, fight deadly creatures and finally get the submarine back in command from the ultimate underwater beast, that red octopus!
But it's not all bad in the underwater depths as he will also come across beautiful landscapes and will acquire many trustworthy allies, in a game that features a compelling and philosophical storyline, unusual visual graphics, classic arcade gameplay with choice difficulty levels and multiple genres in one game!


Whether you're shopping on a budget for something simple, or you're the kind of worker that makes a living in your harness and you only want the best, we've got you covered. For example, the FallTech Contractor+ #7016B Harness is priced very well yet gives you 5-point adjustability for a customized fit. If you need more D-Rings and a belt for construction work, then check out the FallTech Contractor+ #7073B Harness or the Protecta PRO #1161309 Harness. But if you want something more comfortable and with all the bells and whistles, it's hard to beat the FallTech Advanced ComforTech Gel #7081B Harness--premium padding, fully adjustable and a pleasure to wear. But, some would argue that the new DBI-SALA ExoFit NEX Construction Harness is simply the best of breed--the most innovative, easy-to-fit and undeniably cool!
We offer a wide selection of lanyards, from basic to advanced, shock-absorbing or positioning, etc. If you need a "standard" 6 ft lanyard, we recommend you look no further than the FallTech #8240, which comes with standard snap hooks. If you need a large hook on one end, check out the FallTech #82403--or if you need a Y-leg lanyard for 100% tie-off, check out the FallTech #8240Y3.
Beyond standard products like harnesses and lanyards, we carry a full line of anchors, self retracting lifelines, horizontal lifelines and more. Please browse the categories below to see all of our offerings.


As with eclampsia, there are many protocols that the hospital staff must follow to ensure that a baby is doing well during labor and delivery. There are several monitors set up for the baby, and it is critical to pay attention to danger and/or warning signs because there are some things the staff needs to look at to recognize that the baby might be in distress. When doctors step in, they can often prevent damage.
At Steve German, A Law Firm, in Scottsdale, we offer a free consultation in birth injury cases. Call 480-607-9166 to arrange a time to meet with us.
When a mother goes into labor, the unborn baby is carefully monitored. The monitors will allow the doctors and nurses to know if/when the baby is in distress and not getting enough oxygen. There will be an abnormal pattern on the monitor showing that the baby needs help.
At this point, a doctor can perform an emergency C-section, or use other methods to ensure safety. This comes down to hypervigilant attention and foresight.
When babies and mothers are not properly attended, serious and long-term physical and psychological injuries can be sustained, including quadriplegia and retardation.
If your child may have suffered fetal distress at birth, you need to understand why. If there is a failure to recognize fetal distress, it's important that medical professionals are held accountable and you are compensated, so you can take care of your family economically.
We are committed to bringing our comprehensive experience in medical malpractice cases to investigate any circumstance that may have led to fetal distress. We know there's nothing that can undo the damage done in these cases, but we are committed to bringing a compassionate approach to bolster medical accountability and help families economically when these tragic events occur. We encourage you to reach out.
To discuss any Arizona fetal distress issue in a free initial consultation with one of our Scottsdale fetal distress lawyers, call 480-607-9166 or send us an email.


This little company started from making jams and marmalades in my tiny Brooklyn kitchen from the care packages lemons, grapefruit, and rosemary that my family would send me from back home in Louisiana. A nod to my heritage, BRINS takes its name from the Cajun French word for "little bit". A back-to-basics product handcrafted from fresh fruit and spices, BRINS is reflective of my roots in family, hard work, love, and simple joys. I've learned that you can bring family and food with you wherever you go, and BRINS is the "little bit" I've brought to Brooklyn, and am now sharing with you.
We thin-slice whole fresh lemons then slow-simmer with a heavy pinch of fragrant & floral saffron threads. The honey-like scent of saffron hits you first, followed by bright, tart lemon.
Vanilla bean brings a mellowness to round out the boldness of sweet bananas to create this simple but super addictive jam. Peanut butter hasn't seen something this great since chocolate.
Ripe red strawberries are simmered together with bright citrusy & earthy lemongrass for a light twist on everyone's childhood favorite.
Spicy chili peppers are cooked down in a bath of apple cider vinegar with ginger root, fresh peeled garlic and a touch of lime juice. This jam has a smooth lingering spice, great with avocados & cream cheese.
Tart, sweet, local sour cherries and Dona Chai small batch chai tea - need we say more?


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New brewery now open near Centerville
By Mark Fisher
The Dayton area has added another brewery, this one on the well-traveled strip of Miamisburg-Centerville Road (Ohio 725) between Centerville and the Dayton Mall.
Heavier than Air Brewing Co. — located at 497 Miamisburg-Centerville Road in the Normandy Square Shopping Center — opened on Saturday, Aug. 26. "Wheels Up," the aviation-themed brewery said in its Facebook announcement that it had opened its doors to the public.
MORE BEER NEWS: New brewpub next to Dayton Dragons field sets opening date
Initially, Heavier Than Air will be serving primarily other local breweries' offerings as it ramps up its own brewing operations.
SNEAK PEEK: 'Devil Wind' brewery eyes late-2017 opening in Xenia
Brewery co-founder Nick Tarkany Sr. said Heavier Than Air's tap list this week includes one Heavier Than Air beer — an American pale ale — and brews from Dayton Beer Co., Crooked Handle in Springboro, Lucky Star in Miamisburg, Yellow Springs Brewing, Warped Wing in Dayton, and Toxic Brew Co. in Dayton. A selection from Riegele in Dayton's Sister City of Augsburg, Germany is also on the list.
RELATED: Dayton's craft beer: A guide to breweries
The new brewery's hours are 4 p.m. to 9 p.m. Thursday, 4 p.m. to 10 p.m. Friday, 11 a.m. to 10 p.m. Saturday, and noon to 7 p.m. Sunday. An exterior sign for the brewery is coming soon, Tarkany said.
RELATED: New brewery to open this spring east of the Dayton Mall (January 2017)
Heavier Than Air will have a limited food menu and will encourage customers to order delivery or bring food in, Tarkany said.
Tarkany has been an avid home-brewery for more than a decade. The brewery will be overseen by Tarkany and his brother Chris Tarkany, with an assist from two of Nick's sons, Ben and Nick Jr.
RELATED: New Washington Twp. brewery aims for spring opening (April 2017)
For more information or to monitor its progress toward opening, check out the Heavier Than Air Brewing Facebook page.
RELATED: Discounts to begin at Whole Foods today
Springboro pizza restaurant to close tonight
Businesses throughout Dayton area feature Bengals-themed treats
TELL US: How are you celebrating the Bengals heading to the AFC...
Funky fossil: New discovery named after Ohio Players hit
Warped Wing in Springboro to celebrate vintage and barrel-aged beers
Mark Fisher, a 1981 graduate of Ohio State University and a 1976 graduate of Beavercreek High School, is a third-generation Dayton-area resident who has worked for the Dayton Daily News since 1983. He covered higher education and K-12 education accountability issues for nearly 20 years before taking over the food and dining beat in 2006.


Somewhere in the middle of the 2008 season there was a call that challenged the interpretation of the rules. I was looking to verify my understanding, but everywhere I turned the play was through the lens of the team. It was the right call, even though it was controversial. It was the wrong call, and our team got hosed. I wanted to know the how and the why about the ruling, but everyone was focused on the net result of the play and how it impacted the team.
The playoffs approached, and I had a thought about how I could not find a neutral source about the rules and officiating: if it doesn't exist, then create it.
So, I did a soft launch of the Zebra Blog at zebra.benaustro.com — I wasn't even sure how much I was committing to this thing, yet. The first post on Jan. 30, 2009, was about the commissioner's annual press conference in which he alluded to a possible modification to the overtime rules. I also posted a few short blurbs about Super Bowl XLIII, and who knows if anyone saw it. I soon realized I was going to cover the 2009 season because I wanted to, and if a few eyeballs found it interesting, great.
But, whoever had the domain zebrablog.com — probably some zoölogical site, or something — forced me to come up with a creative URL. And early in the 2009 season footballzebras.com was mirroring zebra.benaustro.com. I still kept the Zebra Blog name, but after a while it became FootballZebras.com, and eventually the .com was dropped.
Doing this alone, family balance and mental burnout sometimes left the site without an update for a couple of weeks. I asked Mark Schultz, a moderator of the officiating forum Behind the Football Stripes, if he was interested in writing for the site. Despite the fact that there was no ad revenue to speak of, he said yes quickly. It was then that this site had cured like a freshly poured concrete sidewalk; there was much more than the rules, but officiating in general, and Mark's perspective was instrumental at a pivotal time.
The 2012 labor impasse spiked our viewership, and at the conclusion of that season, we dropped a story that was critical of the selection process of the Super Bowl XLVII crew. Although we did receive fair criticism for taking away from the prestige of a Super Bowl assignment using anonymous sources, those sources wanted to speak out against a system that they felt was broken for some time. The next season, the process was slightly modified. As for Football Zebras, well, there was no turning back.
In the years since Referee magazine added us to their list of websites and apps "worth checking out." In 2015, Sports Illustrated included Football Zebras in their Social 100 list. Somewhere in there, I found time to write a book, and So You Think You Know Football?: The Armchair Ref's Guide to The Official Rules was published in September 2015.
Today, this post is punched out not in some corporate office, but instead on a well-worn laptop with cracks in the plastic while sitting in my living room. On Sundays, I flip around through the games from here, posting updates on the site, and tweeting rules interpretations while being asked by my son to look at his latest Lego creation or with my daughter showing me her dance routine in my "office." That's not to mention a very patient wife who has given up on trying to sync dinner between my writing and Sunday Night Football. The second Sunday in February is her Super Bowl.
In addition, this site has been supported by some very talented writers both past and present: Scott Diller, Cam Filipe, Marcus Griep, Matt Holmquist, Michael Leptic, Josh Lewis, Rich Madrid, David Root, and Patrick Weber. Without your help, this is just my former little side project that never really got off the ground. And thank you, our reader, for setting aside time in your day and for taking an interest in what we write.
I can't wait to see what the next 10 years will bring.
Click on an image to run a slideshow of our history, starting with the very first post.
Congratulations. I enjoy the things you post. I used to enjoy the format. Even though the most recent makeover has made it nearly impossible to follow, I'm still hanging in and I'm glad you are too.
Does anyone else have trouble following the site now? Maybe you can never go back, but I'd sure like to.


Deana Spencer, our current President, has served on the board of BC Pride since 2012, first as Vice President and as President since 2014. She works at PNC Bank as a personal banker since 2015 and is a lifelong Battle Creek resident. She loves spending time with her partner Kim, her three adult children and their partners, and her family.
Deana has served as a Celebrity Server for S.A.F.E. Place's annual fundraiser, on the Rainbow Community Grant Fund steering committee with the Battle Creek Community Foundation, a guest columnist for the Battle Creek Enquirer and the Battle Creek Shopper News, and on the International Summerfest planning committee. Deana is proud to have developed many of the BC Pride programs still in effect today.
Deana is committed and continues to work toward full equity for the LGBTQ+ Community. She is dedicated to making Battle Creek a more diverse and accepting city and embraces its rich history and all it has to offer. Deana will continue to revisit and push the Calhoun County Board of Commissioners to develop an all inclusive EEO Policy that will include LGBTQ+ language.
parents and are in the process of adopting their first son.
Charlie loves volunteering and working towards making sure that everyone in Battle Creek has the same rights. He loves promoting equality and diversity in the city in which he lives. Charlie was a leader in getting the non-discrimination ordinance passed in the City of Battle Creek in 2013.
Charlie also volunteers his time by sitting on the Historic District Commission of Battle Creek and serving as a Board Trustee for the Battle Creek Public School District.
Rebecca Mae, our current secretary, has lived in Battle Creek for 12 years and has served on the board since July 2018. She is the grandmother of five amazing grandchildren, and believes her work on the Pride Board will help ensure a better future for them because she believes that love is love is love is love is love.
Elaine Hadley, our current Treasurer, is a lifelong resident of Battle Creek, retired from Hart, Dole, Inouye Federal Center, and an Army Veteran.
Abi Fukuyama, is a current 3rd year board member. They're very passionate about local government along with trying to get youth more involved. They served 3 years on the Battle Creek Youth Advisory Board (YAB). As well as having served on The Battle Creek Blue Ribbon Advisory Committee. Abi's passion for getting involved began in high school where they served on Lakeview High School's SHIFT (Sexual Health Initiative For Transformation) team and the Reproductive Health Advisory Board.
During their last two years at Lakeview High School, they were the president of the GSA and participated in several teacher trainings on LGBTQ+ issues. They won the Spartan Leader of Students Award their senior year for their LGBTQ+ equity work.
Ashley Feagin, is a white, queer, pansexual, cisgender, southern femme. She is an Assistant Professor at Albion College and teaches computer and lens based art forms. Currently she is serving as the Chair of the Faculty Committee for Diversity for the college where she has gained transferable skills for increasing awareness, diversity and inclusion in organizations.
Ashley joined BC Pride's board in 2018, bringing with her a commitment to the work of diversity and inclusion, as well as, occasionally offering her design skills to visual marketing!
Ashley is also a working artist and has been exhibited internationally. Her work explores the complexity of queer identity and can be viewed on her website (www.ashleyfeagin.com) Ashley lives in Battle Creek with her first child Richard (cat), her only daughter Roxy (dog), her son, Isaac and her spouse Jaimie.
Ian Gallagher, has a background in Criminal Justice and is a former business owner of Homer Entertainment, a video and video game rental store, in Homer, MI. Ian now enjoys his job at Systex in Battle Creek. He moved to Battle Creek in 2009 and has been an active board member since 2013.
Ian is a very strong advocate for HIV/AIDS education and resources and continually strives to promote equality for all LGBTQ+ people in the Battle Creek area.
Jim Haadsma, is the Michigan Democratic State Representative, District 62. Jim also continues to serve as a Worker's Compensation and Social Security SSI attorney with McCroskey Law, P.L.C. since 1987 and is a former District 3 Calhoun County Commissioner.
Jim serves on many non-profit boards and organizations always fighting for justice for the under represented and under served in our community. Jim believes that every person is entitled to the same rights as the next person.
When Jim's four children were young, he would read them the illustrated story, "The Legend of King Christian X of Denmark", about how the King of Denmark stood up to his country's occupiers by declaring that every Dane was entitled to the same rights as the next Dane.
Nancy Kenny, is in her second year as a Battle Creek Pride Board Member and has been an avid BC Pride supporter and volunteer for many years.
"It has been amazing for me to witness, and to be a part of the LGBTQ+ Community here. When I moved to Battle Creek in early 2006, there was no PRIDE DAY or Pride Month. We have, come a long way!" Nancy's favorite event is our Pride Parade and Festival.
Nancy enjoys being active outdoors and spending time with her family when she is not working at Denso Manufacturing in Battle Creek.
Tina Germinder-Metzger, an educator and activist is an active member of the Koinonia Membership at First Congregational Church of Battle Creek. Tina is a lifetime member of Girl Scouts and the leader of her daughter's local Troop 80721.
Tina is called to help others find their voice and seeks opportunity to express her true self. She is continuously learning ways to improve her ability to be a social justice advocate.
She and her wife are parents to two amazing children. Their entire family enjoys being active in several local community organizations.


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Home » Sport » Basketball » NBA
Donovan Mitchell POPS OFF For SEASON-HIGH 43 Points! | December 6, 2022
December 7, 2022 by Sport Desk
NBA published this video item, entitled "Donovan Mitchell POPS OFF For SEASON-HIGH 43 Points! | December 6, 2022" – below is their description.
Never miss a moment with the latest news, trending stories and highlights to bring you closer to your favorite players and teams.
Download now: https://app.link.nba.com/APP22
Led by Donovan Mitchell's season-high 43 points, 6 rebounds, 5 assists, and 4 steals, the Cleveland Cavaliers defeated the Los Angeles Lakers, 116-102.
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