Kidney stone formation is a global health problem with increasing prevalence. Stone formation is a physiochemical process involving crystallization of inorganic salts in the presence of biological constituents in the urinary system. To inhibit kidney stone formation, a better understanding of the underlying physicochemical mechanism of stone formation in the kidney is required. In this thesis, the solubility, nucleation and growth of calcium oxalate (CaOx), the most common inorganic constituent of kidney stones, were studied under different conditions such as ion concentration, pH value, and also the role of inhibitors in water or artificial urine was investigated. The first step towards this work was obtaining the solubility curve of calcium oxalate monohydrate (COM) in the solvent, such as ultrapure water and different buffers, to elucidate the physicochemical conditions which can cause the kidney stone formation (Chapter 2). Beside the solubility study, advanced technology to observe crystal formation in small scale and a very short time was needed. The volume, structure and flow properties inside the kidney inspired us to use microfluidic technology with comparable volume and flow rate. The developed microfluidic devices that mimic pathways in the human kidney were used to study the nucleation and growth of calcium oxalate crystals. The developed devices rendered an alternate perspective to the study of kidney stone formation and showed that microfluidics can provide precise, simple and fast detection of stone formation under various experimental conditions. Initially, the designed microfluidic device allowed us to build a testing platform for the study of nucleation kinetics of CaOx inside isolated environments provided by droplets. Preliminary experiments were performed by dissolving calcium chloride and sodium oxalate in ultrapure water. The aqueous solution, containing the ions, forms the droplet phase and oil were used as the continuous phase. Altering the pH values, as well as increasing the concentration of additives such as magnesium and osteopontin (OPN), were shown to slow down the nucleation kinetics, or even inhibit nucleation (Chapter 3). @en

The increasing prevalence of urolithiasis in industrialized societies triggered considerable interest in how various species found in urine regulate the nucleation and growth of common kidney stone constituents such as calcium oxalate (CaOx). Yet, the role macromolecules play in kidney stone formation is often overlooked due to their low concentration in urine. In this study, we investigate the nucleation kinetics of CaOx in artificial urine with droplet-based microfluidic induction time measurements at varying concentrations of oxalate and hyaluronic acid (HA), a polysaccharide commonly found in urine. The formation of two pseudo-polymorphic forms of calcium oxalate crystals, calcium oxalate monohydrate (COM) and calcium oxalate dihydrate (COD), are carefully monitored using polarized light microscopy in induction time experiments. COM and COD nucleated concomitantly in artificial urine yet with distinct kinetics. Our results indicate that higher oxalate concentrations favor the formation of COD, the metastable form, over COM, the most stable form. Moreover, COD is also the fastest nucleating form in droplets under studied conditions. Furthermore, increasing the concentration of HA at fixed calcium and oxalate concentrations favored the nucleation of COM. We observed that in droplets where COM nucleated first, COD was not formed within the experimental time scale. However, in the droplets where COD appeared first, COM crystals were also observed later. We hope our findings shed light on the role macromolecules such as HA plays in dictating the pseudo-polymorphic form of CaOx and guide next generation treatments.@en

Detected kidney stone cases are increasing globally, yet knowledge on the conditions for stone formation is lacking. Experimental approaches mimicking the micro-environmental conditions present in vivo can help sci-entists untangle intertwined physiochemical and biological phenomena leading to kidney stone formation. As crystal nucleation often initiates at liquid-solid interfaces, the interface morphology plays a significant role in the rate of nucleation. Within the nephron, the functional unit of the kidney, four segments can be distinguished that contain different surface morphologies. Particularly, the cells lining these segments contain protrusions in the shape of nanopillars that vary in length, diameter and spacing. Exploiting the opportunities provided by organ- on-a-chip technology, we designed and manufactured a proof-of-principle microfluidic device proposed to in-crease our understanding of the relation between kidney surface morphology and kidney stone crystallization. We used two-photon polymerization to fabricate biocompatible surfaces that mimic the nephron morphologies with materials properties similar to those of biological structures. The fabricated cilia were incorporated in the microfluidic device, which was designed to observe in vitro crystallization of calcium oxalate under flow@en

Selective crystallization of polymorphs is highly sought after in industrial practice. Yet, state-of-the-art techniques either use laboriously engineered solid surfaces or strenuously prepared heteronucleants. We propose an approach where surfactants in solution self-assemble effortlessly into mesoscopic structures dictating the polymorphic outcome of the target solute. Sodium dodecyl sulfate (SDS) surfactant is used as a tailored additive to crystallize different polymorphic forms of a model active pharmaceutical ingredient, d-mannitol. Different mesoscopic phases of SDS template particular polymorphs: packed monolayers, micelles, and crystals favored the β, α, and δ forms of d-mannitol, respectively. A synergistic effect of topological templating and molecular interactions is proposed as the rationale behind the observed selective crystallization of polymorphs. This crystal engineering technique suggests that surfactant self-assemblies can be used as tailored templates for polymorphic control.@en

A droplet-based microfluidic platform is presented to study the nucleation kinetics of calcium oxalate monohydrate (COM), the most common constituent of kidney stones, while carefully monitoring the pseudo-polymorphic transitions. The precipitation kinetics of COM is studied as a function of supersaturation and pH as well as in the presence of inhibitors of stone formation, magnesium ions (Mg2+), and osteopontin (OPN). We rationalize the trends observed in the measured nucleation rates leveraging a solution chemistry model validated using isothermal solubility measurements. In equimolar calcium and oxalate ion concentrations with different buffer solutions, dramatically slower kinetics is observed at pH 6.0 compared to pHs 3.6 and 8.6. The addition of both Mg2+ and OPN to the solution slows down kinetics appreciably. Interestingly, complete nucleation inhibition is observed at significantly lower OPN, namely, 3.2 × 10-8 M, than Mg2+ concentrations, 0.875 × 10-4 M. The observed inhibition effect of OPN emphasizes the often-overlooked role of macromolecules on COM nucleation due to their low concentration presence in urine. Moreover, analysis of growth rates calculated from observed lag times suggests that inhibition in the presence of Mg2+ cannot be explained solely on altered supersaturation. The presented study highlights the potential of microfluidics in overcoming a major challenge in nephrolithiasis research, the overwhelming physiochemical complexity of urine.

@en

Hypothesis: Our ability to dictate the colloid geometry is intimately related to self-assembly. The synthesis of anisotropic colloidal particles is currently dominated by wet chemistry and lithographic techniques. The wet chemical synthesis offers limited particle geometries at bulk quantities. Lithographic techniques, on the other hand, provide precise control over the particle shape, although at lower yields. In this respect, two-photon polymerization (2PP)¹ has attracted growing attention due to its ability to automatically fabricate complex micro/nano structures with high resolution. Experiments: We manufacture precisely designed colloids with sizes ranging from 1 µm to 10 µm with 2PP and optimize the process parameters for each dimension. Moreover, we study the shape dependent Brownian motion of these particles with video microscopy and estimate their diffusion coefficients. Findings: We observe that increasing the geometrical anisotropy leads to a pronounced deviation from the analytically predicted diffusion coefficient for disks with a given aspect ratio. The deviation is attributed to stronger hydrodynamic coupling with increasing anisotropy. We demonstrate, for the first time, 2PP manufacturing of colloids with tailored geometry. This study opens synthesis of colloidal building blocks to a broader audience with limited access to cleanrooms or wet-chemistry know-how.

@en

Accurate Calcium Oxalate Monohydrate (COM) solubility measurements are essential for elucidating the physiochemical mechanism behind the formation of kidney stones, nephrolithiasis. Yet the reported solubility values of COM in ultrapure water, arguably the simplest solvent relevant for nephrolithiasis, vary significantly depending on implemented method. To address this variation, we present an experimental study of the solubility of COM validated by a model based on the Debye–Hückel theory describing the solution chemistry and the complex formation. We also carefully monitor potential pseudopolymorphic/hydrate transitions during the solubility measurements with in-situ and ex-situ methods. Our results indicate that the solubility of COM in ultrapure water is a weak function of temperature. However, the measured solubility varies significantly in buffer solutions across physiologically relevant pH values at body temperature. The proposed model explains observed trends as a combined effect of ionic strength, protonation reactions, and soluble complex formation. Moreover, it predicts solubility of COM in buffer solutions remarkably well using our measurements in ultrapure water as input, demonstrating the consistency of presented approach. The presented study parleying experiments and modelling provides a solid stepping stone to extend the physiochemical understanding of nephrolithiasis to more realistic solutions laden with biological complexity.@en

Ventilator-associated pneumonia (VAP) is a type of hospital-acquired infection with high mortality and morbidity rates that is seen in critically ill patients who are connected to mechanical ventilators in intensive care units. Conventional infection control measures may remain insufficient for control and prevention of VAP. In the present study, biofilms of most common pathogens, which are responsible for VAP, namely Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, and Candida albicans, were grown in the lumen of endotracheal tubes (ETs) under low shear to mimic the biofilm formation in clinical settings using the drip flow reactor system. Antibiofilm efficacies of two different plasma treatment methods were tested on the biofilms that were grown in ETs. In the first method, deionized water (DIW), phosphate-buffered saline (PBS) solution, and N-acetylcysteine (NAC) solution were treated with air dielectric barrier discharge (DBD) plasma and then nebulized on the biofilms. In the second method, air plasma afterglow (APA), which was generated using a DBD jet electrode, was flown through the lumen of ETs. Effects of two different plasma treatment methods on biofilms were evaluated using colony-counting and XTT assays. The quantity of extracellular polymeric substance (EPS) was evaluated with the safranin assay. Scanning electron microscopy (SEM) was used to visualize the effect of various plasma treatment methods on biofilms. Cytotoxic effects of two different plasma treatment methods were tested on the human tracheal epithelial cell line. Our results suggest that the nebulized plasma-treated NAC solution had a superior antibiofilm effect as compared with DIW and PBS. Two different plasma treatment methods used effectively inactivated biofilms and significantly reduced the quantity of EPS without a significant damage to the human tracheal epithelial cell line. Furthermore, SEM images showed the inactivation of microbial cells mainly through membrane damage.@en

Objective: The aim of the present, microcomputed tomographic (mCT) and histological study, was to evaluate the effect of surface modification by atmospheric pressure cold plasma (APCP) on vertical guided bone regeneration in a rabbit calvaria model. Material-Methods: The experimental study was conducted on 12 male New Zealand rabbits with healing periods of 45 and 90 days. Following surgical exposure of the calvarium, 4 customized titanium cylindricalders were fixed. Surface modification was achieved by application of APCP on 2 of cylinders (Pþ) in each calvarium and other cylinders were set as control (P-). In both experimental and control groups, one of the cylinders was filled with bone graft (Gþ) while the other one was left empty (G-). To evaluate short term effects, randomly selected 6 animals were sacrificed at the end of 45 days and remaining 6 animals were left for observing long term effects. Histological and mCT evaluations were used to examine new bone formation. Results: In mCT imaging; the bone volume was greater (P < 0.05) in grafted groups than nongrafted groups in both short and long term. The bone height values were significantly different in (P-G-) group than other groups (P < 0.05) in both evaluation periods. The histological evaluations revealed significant differences between PþGþ group and other groups but in long term both plasma treated groups revealed more bone formation than non plasma treated groups. Conclusion: Modification of the surfaces of titanium cylinders by APCP treatment, accelerated the bone regeneration either bone graft used or not in a rabbit calvaria model.@en

The bleaching efficacy of common bleaching agents and deionized water treated with non-thermal atmospheric pressure plasma in the pulp chamber for nonvital tooth bleaching was evaluated. A total of 120 extracted human maxillary first incisors were stained using human blood. Teeth were randomly divided into eight groups (n = 15). In the first four groups, teeth were bleached using 35% hydrogen peroxide gel, 37% carbamide peroxide gel, 2:1 (w/v) sodium perborate paste, and deionized water for 30 min. In the remaining groups, bleaching agents were treated with non-thermal atmospheric plasma for 5 min inside the pulp chamber. Overall color changes (∆E) were determined using Commission Internationale de L’Eclairage Lab Colour System. The plasma-assisted tooth bleaching has not increased tooth temperature beyond 37°C. Bleaching efficacies of bleaching agents were significantly improved when treated with non-thermal atmospheric plasma compared to their application (P < 0.05). A remarkable bleaching effect was obtained when bleaching agents were substituted with water and when treated with non-thermal atmospheric plasma. Non-thermal atmospheric plasma treatment could be a novel tool for activation of bleaching agents in the pulp chamber for nonvital tooth bleaching procedure. Moreover, water could be used as a novel bleaching agent when treated with the non-thermal atmospheric plasma to eliminate possible risks which might arise from peroxide-containing agents.

@en

The purpose of this in vitro study was to evaluate the effect of the non-thermal plasma (NTP) treatment on the wettability and surface roughness of different types of CAD/CAM materials as well as the shear bond strength (SBS) of adhesive resin cement to the treated surfaces. Three different materials, namely; resin nano ceramic, feldspathic ceramic and poly(methyl methacrylate) (PMMA)-based samples were treated with NTP for different time points to evaluate the effect of NTP treatment on the surface properties of CAD/CAM materials. Moreover, surfaces of CAD/CAM materials were visualised with scanning electron microscopy (SEM). A 90-second NTP treatment time was determined as the optimum time for the highest measured wettability, and thus, the 90-second NTP treated samples were used for the SBS test and evaluation of the failure types. Our results revealed the NTP treatment lowered the contact angles and increased the roughness of all tested materials. Moreover, The NTP treatment significantly enhanced the SBS of resin nano ceramic and feldspathic ceramic-based materials. NTP could be considered as a novel pre-treatment method to improve surface properties and the bonding performance of ceramic-based CAD/CAM materials.@en

We detail the analysis of centrifugal homogenization process by a hydrodynamic model and the model-guided design of a low-cost centrifugal homogenizer. During operation, centrifugal force pushes a multiphase solution to be homogenized through a thin nozzle, consequently homogenizing its contents. We demonstrate and assess the homogenization of coarse emulsions into relatively monodisperse emulsions, as well as the application of centrifugal homogenization in the mechanical lysis of mpkCCD mouse kidney cells. To gain insight into the homogenization mechanism, we investigate the dependence of emulsion droplet size on geometrical parameters, centrifugal acceleration, and dispersed phase viscosity. Our experimental results are in qualitative agreement with models predicting the droplet size. Furthermore, they indicate that high shear rates kept constant throughout operation produce more monodisperse droplets. We show this ideal homogenization condition can be realized through hydrodynamic model-guided design minimizing transient effects inherent to centrifugal homogenization. Moreover, we achieved power densities comparable to commercial homogenizers by model guided optimization of homogenizer design and experimental conditions. Centrifugal homogenization using the proposed homogenizer design thus offers a low-cost alternative to existing technologies as it is constructed from off-the-shelf parts (Falcon tubes, syringe, needles) and used with a centrifuge, readily available in standard laboratory environment.

@en

The ongoing search for better antibacterial wound care dressings has led to the design and fabrication of advanced functional nanomaterials. Taking advantage of electrospinning and cold atmospheric plasma (CAP), free-standing nanofibrous scaffolds are promising for use in novel biomedical applications. Random and aligned polyvinyl alcohol (PVA)/polyacrylic acid (PAA) nanofiber scaffolds are fabricated by electrospinning and treated with CAP. In this study, we investigate the effects of CAP treatment on alignment, hydrophilicity, antibacterial activity, and biocompatibility in determining the surface properties of the nanofibrous scaffolds. The results of vibrational polarization spectroscopy analysis indicate that CAP treatment changes the degree of alignment of the nanofibers. Furthermore, both random and aligned CAP-treated nanofibrous scaffolds show significant antibacterial activity against the E. coli strain. The results of an in vitro scratch assay reveal that CAP treatment of PVA/PAA nanofibers has no toxic effect.@en

This in vitro study aimed to evaluate the effect of nonthermal atmospheric pressure plasma (NAPP)-activated hydrogen peroxide (HP) and deionized water (DW) on enamel microhardness and enamel surface morphology. @en

The aim of the present study was to compare antibiofilm efficacies of the laser in contact and noncontact application modes and cold atmospheric plasma (CAP) on Staphylococcus aureus biofilm grown on sandblasted, large grit, acid-etched (SLA) titanium discs as an in vitro model of biofilm eradication on dental implant materials. Methods: S. aureus biofilm was matured on titanium discs for 7 days then, treated with contact and noncontact Er:YAG laser and CAP. Antibiofilm efficacy of laser and plasma treatments were evaluated with colony counting and safranin assays. Surface characteristics of titanium disc were analyzed with scanning electron microscopy and surface roughness measurements. Temperature distribution over titanium discs were presented for the thermal safety assessment of laser and plasma treatments@en

Surgical site infections have a remarkable impact on morbidity, extended hospitalization and mortality. Sutures strongly contribute to development of surgical site infections as they are considered foreign material in the human body. Sutures serve as excellent surfaces for microbial adherence and subsequent colonization, biofilm formation and infection on the site of a surgery. Various antimicrobial sutures have been developed to prevent suture-mediated surgical site infection. However, depending on the site of surgery, antimicrobial sutures may remain ineffective, and antimicrobial agents on them might have drawbacks. Plasma, defined as the fourth state of matter, composed of ionized gas, reactive oxygen and nitrogen species, free radical and neutrals, draws attention for the control and prevention of hospital-acquired infections due to its excellent antimicrobial activities. In the present study, the efficacy of non-thermal atmospheric plasma treatment for prevention of surgical site infections was investigated. First, contaminated poly (glycolic-co-lactic acid), polyglycolic acid, polydioxanone and poly (glycolic acid-co-caprolactone) sutures were treated with non-thermal atmospheric plasma to eradicate contaminating bacteria like Staphylococcus aureus and Escherichia coli. Moreover, sutures were pre-treated with non-thermal atmospheric plasma and then exposed to S. aureus and E. coli. Our results revealed that non-thermal atmospheric plasma treatment effectively eradicates contaminating bacteria on sutures, and non-thermal atmospheric plasma pre-treatment effectively prevents bacterial colonization on sutures without altering their mechanical properties. Chemical characterization of sutures was performed with FT-IR and XPS and results showed that non-thermal atmospheric plasma treatment substantially increased the hydrophilicity of sutures which might be the primary mechanism for the prevention of bacterial colonization. In conclusion, plasma-treated sutures could be considered as novel alternative materials for the control and prevention of surgical site infections.@en

This in vitro study aimed to evaluate the bleaching efficacy and color stability of nonthermal atmospheric pressure plasma (NAPP)-activated 40% hydrogen peroxide (HP) and deionized water (DW). Methods: Two hundred-forty bovine enamel−dentin samples were stained with a coffee solution before bleaching. Samples were randomly divided into 12 (n = 20): (1) HP for 5 minute (HP-5), (2) HP-10, (3) HP-20, (4) Plasma-HP-5, (5) P-HP-10, (6) P-HP-5/HP-15, (7) P-HP-10/HP-10, (8) P-DW-5, (9) P-DW-10, (10) DW-5, (11) DW-10, and (12) DW-20. After bleaching, the samples were re-stained for 48 h using red wine. The color differences measured using a dental spectrophotometer after bleaching and after re-staining were reported as ΔE1 and ΔE2, respectively. The data were analyzed using Kruskal−Wallis and Mann−Whitney U tests (p< 0.05).@en

INTRODUCTION: Barrier membranes are used to create an empty space in the bone defects and to stabilize the blood clot to prevent soft tissue migration. Materials used as barriers should be characterized by some physico-chemical properties to improve biocompatibility, tissue integration, void formation ability and clinical use. The application of atmospheric cold plasma(ACP), which has been shown to increase surface energy and wettability in various materials intended to work, angularly examines and correlates the cellular uptake, protein adsorption and wettability on the surface of the titanium mesh. METHODS: Mesh with dimensions of 1x1 cm and a diameter of 1.6 mm with a thickness of 0.3 mm was treated with air barrier plasma obtained by using a specially produced microsensor pulsed plasma power source. While there is no application in the control group, the mills in the study group were subjected to ACP application with microgeneration plasma generator for 90 seconds. The contact angle measurements were made using a goniometer. RESULTS: The best result in the wettability evaluation was found to be measured in 90 seconds time. There was no statistically significant difference between the amount of protein in samples removed from the incubator after 6 hours of incubation period (p>0.05). There is a statistically significant difference between the groups in the 24-hour time frame (p<0.05). No significant difference was found on day 1 in cell viability (p>0.05) and expression, but a significant difference was found on days 3 and 5 (p<0.05). DISCUSSION AND CONCLUSION: Recently, argon atmospheric pressure plasma has been reported to increase cell diffusion and wettability in titanium discs. In terms of wettability, in accordance with other studies, our work also resulted in the ACP application increasing the wettability and decreasing the contact angle. Long-term effects on cell adhesion and proliferation are seen. Consequently, there is a need for further experimental and clinical studies of the subject. @en

Antimicrobial activities of non-thermal atmospheric plasma-treated liquids on various bacterial and fungal strains in their planktonic and biofilm forms have been widely reported. Since most of the plasma-treated liquids are water based, they might be washed off certain surfaces and they cannot be applied for the infections of respiratory tract. In the present study we have tested antimicrobial activities of plasma-treated N-acetyl cysteine solution (NAC), phosphate buffered saline (PBS) solution and deionized water when nebulized over planktonic forms of E. coli and S. aureus. Methods. Antimicrobial activities of nebulized plasma-treated liquids were evaluated with zone of inhibition test and colony counting assay. Moreover pH of NAC, PBS solution and deionized water were measured before, after plasma treatment and during nebulization since low pH is well known consequence observed in plasma-treated liquids. Results. Our results have revealed that pH of plasma-treated NAC, PBS solution and DIW decreases after plasma treatment consistent with previous reports and does not change during nebulization. Moreover, antimicrobial activity assessment indicates that nebulized plasma-treated NAC shows the strongest antimicrobial activity, which leads complete inactivation of bacteria for 103 to 106 CFU/ml initial bacterial load and 5-log reduction for 107 CFU/ml initial bacterial load on both E. coli and S. aureus. Conclusions. Plasma-treated liquids could retain their antimicrobial activity during nebulization and nebulization could be considered as a future alternative method for delivery of plasma-treated liquids for respiratory tract infections. @en

The aim of this study was to investigate the synergistic effect of cold atmospheric plasma (CAP) treatment and RGD peptide coating for enhancing cellular attachment and proliferation over titanium (Ti) surfaces. The surface structure of CAP-treated and RGD peptide-coated Ti discs were characterized by contact angle goniometer and atomic force microscopy. The effect of such surface modification on human bone marrow derived mesenchymal stem cells (hMSCs) adhesion and proliferation was assessed by cell proliferation and DNA content assays. Besides, hMSCs’ adhesion and morphology on surface modified Ti discs were observed via fluorescent and scanning electron microscopy. RGD peptide coating following CAP treatment significantly enhanced cellular adhesion and proliferation among untreated, CAP-treated and RGD peptide-coated Ti discs. The treatment of Ti surfaces with CAP may contribute to improved RGD peptide coating, which enables increased cellular integrations with the Ti surfaces.@en