<p>Najče&scaron;ća komplikacija nakon ugradnje silikonskih implantata za dojku je kontraktura fibrozne kapsule (KK), koja se normalno stvara oko implantata u sklopu reakcije oko stranog tela. Najozbiljnija komplikacija nakon ugradnje silikonskih implantata za dojku je anaplastični krupnoćelijski limfom koji se javlja isključivo kod pacijentkinja koje imaju ugraĎene implantate (eng. Breast-implant associated anaplastic large cell lymphoma &ndash; BIA ALCL). Uzrok nastanka ovih komplikacija ostaje nepoznat. Ustanovljeno je da se KK manje javlja kod implantata koji imaju makroteksturisanu povr&scaron;inu i kod onih koji su presvučeni poliuretanskom penom. S druge strane, BIA-ALCL se če&scaron;će dijagnostikuje kod pacijentkinja kojima su ugraĎeni upravo makroteksturisani implantati. Subklinička infekcija koja predstavlja odgovor organizma na postojanje biofilma na ugraĎenim implantatima, predstavlja jedan od najznačajnijih etiolo&scaron;kih faktora za nastanak KK i BIA-ALCL. Biofilm je konglomerat mirkoorganizama uronjenih u matriks koji ih &scaron;titi od dejstva antibiotika i antiseptika. Kako je nemoguće delovati medikamentozno na eradikaciju biofilma, brojni autori daju razne preporuke u cilju izbegavanja kontaminacije implantata tokom operativnog zahvata, a time i formiranja biofilma. Pored brojnih mera, savetuje se i ispiranje džepa u koji će se plasirati proteza kao i same proteze, nekim od antiseptičkih ili antibiotskih rastvora. Do sada ne postoje prihvaćene jasne preporuke o načinu ispiranja različitih implantata, objavljena su samo lična iskustva raznih autora. Ciljevi ovog istraživanja su bili da se ustanovi mogućnost formiranja biofilma četiri različite bakterije (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa i Ralstonia pickettii) na tri različito teksturisana silikonska implantata za dojku (sa porama veličine 70-150 &mu;m, 50&ndash;900 &mu;m i 13 &mu;m) u in vitro uslovima; da se ispita da li ispiranje antisepticima (oktenidindihidrohloridom i povidon jodom), ili antibiotikom (cefuroksimom) ili istovremeno me&scaron;avinom povidon joda i antibiotika pre bakterijske kontaminacije sa četiri različite bakterije ima uticaja na formiranje biofilma na tri različito teksturisana implantata za dojke u in vitro uslovima; i da se ispita efekat antiseptika u odnosu na efekat antibiotika na formiranje bakterijskog biofilma na tri različito teksturisana silikonska implantata za dojku. Istraživanje je koncipirano kao prospektivna studija u vidu eksperimenta koji je izveden u Laboratoriji za mikrobiologiju, Instituta za javno zdravlje Vojvodine u Novom Sadu. Za izvoĎenje eksperimenta kori&scaron;ćeni su uzorci tri vrste silikonskih implantata za dojku sa različito teksturisanom povr&scaron;inom, odnosno porama različite veličine: 70-150 &mu;m, 50&ndash;900 &mu;m, i 13 &mu;m. Od svakog od navedenih implantata su pravljeni uzorci, sečenjem kapsula implantata na komadiće veličine 1x1 cm. Ukupno je bilo 1440 uzoraka. Na osnovu teksture uzorci su podeljeni u tri grupe: Grupa 1 (pore veličine 70-150 &mu;m), Grupa 2 (pore veličine 50&ndash;900 &mu;m) i Grupa 3 (pore veličine 13 &mu;m). Svaka od ovih grupa je dalje podeljena u jednu kontrolnu grupu i po četiri ispitivane grupe. Nakon sterilizacije uzoraka svaka kontrolna grupa je kontaminirana sa po 100&mu;l bakterijskog bujona Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) i Ralstonia pickettii (n=30). Ispitivane grupe se bile podeljene prema načinima ispiranja na one u kojima su uzorci prvo ispirani: oktenidin &ndash; dihidrohloridom ili povidon jodom ili cefuroksimom ili kombinacijom povidon joda i dva antibiotika, pa potom kontaminirani sa po 100&mu;l bakterijskog bujona Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) i Ralstonia pickettii (n=30). Po zavr&scaron;enoj kontaminaciji, uzorci su se inkubirali na temperaturi od 37&deg;C u trajanju od 96h, čime su stvoreni uslovi za formiranje biofilma. Nakon inkubacije, svaki pojedinačni uzorak je uronjen u sterilan tripton soja bujon, izlagan soničnoj energiji u trajanju od 1minuta i zatim vorteksiran 1 minut, čime je omogućeno odvajanje nastalog biofilma od implantata. Za ispitivanje sposobnosti formiranja biofilma kori&scaron;ćena je modifikovana tehnika sa mikrotitar pločom po Stepanoviću. Rezultati su pokazali da sve četiri ispitivane bakterije S. epidermidis, S. aureus, P. aeruginosa i Ralstonia pickettii statistički značajno vi&scaron;e stvaraju biofilm na implantatima sa porama veličine 50&ndash;900 &mu;m u odnosu na pore 70-150 &mu;m i u odnosu na pore veličine 13 &mu;m. Biofilm se statistički značajno vi&scaron;e stvara na porama veličine 70-150 &mu;m u odnosu na pore 13 &mu;m. Jedini izuzetak je Pseudomonas aeruginosa kod kojeg ne postoji statistični značajna razlika u produkciji biofilma na teksturisanim implantatima sa porama veličine 70-150 &mu;m u odnosu na one sa porama 13 &mu;m. TakoĎe, sve četiri ispitivane bakterije statistički značajano manje stvaraju biofilm nakon ispiranja povidon jodom, oktenidin-dihidrohloridom ili rastvorom antibiotika u sve tri grupe implantata, u odnosu na povr&scaron;ine koje nisu ispirane. Izuzetak je S. epidermidis u Grupi 3 kod kojeg nije utvrĎeno statistički značajno manje formiranje biofilma nakon ispiranja oktenidin dihidrohloridom u odnosu na neispiranje. Cefuroksim je bio efikasniji u sprečavanju formiranja biofilma sve četiri ispitivane bakterije u odnosu na neispiranje u Grupi 1, kao i za S. epidermidis i Ralstoniu Pickettii u Grupi 2. Cefuroksim se nije pokazao statistički značajno efikasnim u sprečavanju formiranja biofilma S. aureus i P. aeruginosa u Grupi 2, kao ni kod jedne bakterije u Grupi 3. Dalje je dokazano da su antiseptici (oktenidin-dihirohlorid i povidon jod) kao i me&scaron;avina povidon joda i dva antibiotika (cefuroksim i gentamicin), statistički značajno efikasnji od ispiranja samo antibiotikomcefuroksimom u smanjenju formiranja biofilma sve četiri ispitivane bakterije kod sva tri ispitivana, različito teksturisana silikonska implantata. Rezultati su pokazali da je ispiranje povidon jodom statistički značajno efikasnije u prevenciji stvaranja biofilma kod skoro svih ispitivanih bakterija od ispiranja oktenidin- dihidrohloridom u sve tri grupe implantata. Statistički značajna razlika nije utvrĎena u prevenciji stvaranja biofilma Staphylococcus aureusa kod sve tri grupe implantata prilikom ispiranja povidon jodom u odnosu na oktenidin- dihidrohlorid, kao i kod Ralsotnia pickettii u Grupi 2. Na osnovu rezultata ove studije, preporuka je da se koriste mikroteksturisani implantati kao i da se oni, pre ugradnje isperu povidon jodom ili me&scaron;avinom povidon jod i dva antibiotika (cefuroksim i gentamicin), u cilju prevencije stvaranja biofilma, a time i postoperativnih komplikacija koje mogu nastati nakon ugradnje implantata.</p> / <p>The most common complication after breast implant surgery is contracture of capsule, which is normally formed around implants as part of foreign body reaction. The most sincere complication after this kind of surgery is breast implant associated anaplastic large cell lymphoma (BIA-ALCL). The cause of these complications is still unknown. It is evident that capsular contracture (CC) is seen less frequently in patients with macro-textured implants and in those with implants covered with polyurethane foam. On the other hand, BIA-ALCL is diagnosed more frequently in patients with those, macro-textured implants. Subclinical infection, defined as an response of organism on presence of biofilm on the implant, is considered to be one of the most important etiologic factors for CC and BIA-ALCL. Biofilm is a conglomerate of microorganisms immersed into matrix, which protects them from influence of antibiotics and antiseptics. As it is impossible to eradicate biofilms with medicaments, many authors suggest different steps in order to avoid contamination of the implant during the operation and therefore, prevent the formation of biofilm. Among many tips, it is recommended to irrigate the pocket for breast implant and the implant itself, with some antiseptic or antibiotic solution. Up till now, there is no agreed consensus on the type of irrigation for different implants. Only personal experiences of a few authors have been published. Aims of this research were: to establish the possibility of biofilm formation of four different bacteria (Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa and Ralstonia pickettii) on three differently textured breast implants (with pore diameter of 70-150 &mu;m, 50&ndash;900 &mu;m and 13 &mu;m) in vitro; to examine whether the irrigation of implant with antiseptics (povidone iodine and octenidine dihydrochloride), antibiotics (cefuroxime) or mixture of povidone iodine and two antibiotics, before the contamination with bacteria, has an influence on the incidence on biofilm formation on three differently textured implants; and to examine the effect of antiseptics in contrast to the effect of antibiotics on biofilm formation on three differently textured breast implants. The study was conducted as a prospective research that took place at the Laboratory for microbiology, at the Institute of public health of Vojvodina in Novi Sad. For the experiment, three types of silicone breast implants were used with different pore sizes: 70-150 &mu;m, 50&ndash;900 &mu;m and 13 &mu;m. Samples were made by cutting each of these types of implants into pieces sized 1x1cm. There were 1440 samples in total. According to texture, samples were divided it three groups: Group 1 (pore size 70-150 &mu;m), Group 2 (pore size 50&ndash;900 &mu;m) and Group 3 (pore size 13 &mu;m). Furthermore, each of these groups was divided in one control and four test groups. After sterilisation of samples, every control group was contaminated with 100&mu;l of bacterial broth of Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) and Ralstonia pickettii (n=30). Tested groups were divided according to type of irrigation into those where samples were firstly irrigated with either: octenidine dihydrochloride of povidone iodine or cefuroxime of mixture of povidone iodine with two antibiotics, and after the irrigation, contaminated with 100&mu;l bacterial broth of Staphylococcus epidermidis (n=30), Staphylococcus aureus (n=30), Pseudomonas aeruginosa (n=30) and Ralstonia pickettii (n=30). After contamination, samples were incubated on 37&deg;C for 96h, which created excellent conditions for biofilm formation. After incubation, each sample was dipped into sterile tripton soy broth, and then exposed to sonic energy for 1 minute and vortexed for 1 minute, which made biofilm separate from the implant. For testing the capability of biofilm formation, modified technique with microtitar plates described by Stepanović was used. Results show that all four examined bacteria S. epidermidis, S. aureus, P. aeruginosa and Ralstonia pickettii form more biofilm on implants with pore sizes 50&ndash;900 &mu;m compared to implants with pore size 70-150 &mu;m and those with 13 &mu;m. Statistical significance was found in biofilm formation on implants with pores 70-150 &mu;m compared to implants with pores 13 &mu;m. Furthermore, all four examined bacteria form statistically less biofilm after the irrigation with any of used solutions: povidone iodine, octenidine dihydrochloride, antibiotic solution of mixture of povidone iodine and two antibiotics, in all three groups of implants compared to surfaces that were not irrigated. The exception is S. epidermidis in Group 3, where no statistical significance was found on biofilm formation after the irrigation with octenidine dihydrochloride compared to non-irrigation. Cefuroxime was more efficient in biofilm prevention for all four tested bacteria compared to non-irrigation in Group 1 and for S. epidermidis and Ralstonia pickettii in Group 2. There was no statistical significance found in prevention of S. aureus i P. aeruginosa biofilms when irrigating with cefuroxime in Group 2, as well as for all tested bacteria in Group 3. Furthermore, it was verified that antiseptics (octenidin dihydrochloride and povidone iodine) and mixture of povidone iodine and two antibiotics (cefuroxime and gentamycin), were statistically more efficient in biofilm prevention of all four examined bacteria in all groups of implants, compared to irrigation with antibiotic-cefuroxime alone. Results show that irrigation with povidone iodine is statistically more efficient in biofilm prevention of almost all examined bacteria compared to irrigation with octenidine dihydrochloride in all groups of implants. There was not found any statistical significance in prevention of Staphylococcus aureus biofilm when irrigating with povidone iodine compared to octenidine dihydrochloride in all groups of implants, and also in biofilm prevention of Ralsotnia pickettii in Group 2. According to results of this research, it is recommended to use micro-textured implants and to irrigate them with povidone iodine or mixture of povidone iodine and two antibiotics (cefuroxime and gentamycin) prior the implementation, in order to prevent biofilm formation which is most probable cause of postoperative complications after implant surgery.</p>

The issue of prosthetic infection leading to implant failure due to the formation of bacterial biofilms on biomaterial surfaces has been widely recognized as a major issue, often leading to revision surgery. The growing number of patients requiring synthetic biomaterials as implants is on the rise and so is the risk of infection arising from pre/peri-/post-operative surgical procedures. Traditional antibiotic treatment has led to the emergence of bacterial drug resistance. Therefore, the development of novel bactericidal methods to combat drug resistant microbial pathogens is the need of the hour. The first part of the thesis is an attempt to address prosthetic infection by the development of novel ultrasmall gold nanoparticles (AuNPs) which are cytocompatible and present a therapeutic dosage window for eliciting antimicrobial property. Towards this end, ultrasmall AuNPs with 0.8 nm and 1.4 nm gold core sizes, stabilized by monosulphonated triphenylphosphine ligand shells were synthesized. Such intricately designed AuNPs with ultrasmall gold cores and phosphine-based ligand chemistry were demonstrated to be highly potent bactericidal agents against staphylococci, the most common human pathogen causing biomaterial associated infection. The antibacterial efficacy of these AuNPs was significant even in mature staphylococcal biofilms. In another study, the application of high strength pulse magnetic fields (1-4 Tesla) was examined for bacterial growth inactivation in vitro. A magnetic field strength dependent decrease in bacterial viability with a concomitant increase in the production of reactive oxygen species (ROS) and longer doubling times were recorded. The mechanism of action was explained through an analytical model which involves ion-transport interference of essential ions like Ca2+ and Mg2+ and disruption of FeS clusters leading to inactivation of bacterial redox enzymes. On the contrary, such high magnetic fields did not pose any detrimental effects to eukaryotic cells under similar exposure. Additionally, the potency of low intensity direct current electric field (DC EF: 1V/cm) against biofilm formation by methicillin resistant Staphylococcus aureus (MRSA) was explored on antimicrobial surfaces of hydroxyapatite and Zinc oxide (HA-xZnO; x = 0, 5, 7.5 and 10 wt%). An EF exposure time dependent decline in the viability and stability of MRSA biofilms were noted. Further, EF treatment resulted in bacterial membrane depolarization and reduced biofilm formation on HA-ZnO composites, independent of the substrate composition. In summary, the above three studies were cases of the developmental methods to address prothetic infection. The second part of the thesis is focused on the development of magneto-responsive biomaterials as implants for orthopaedic applications. Under this category, the sintering/ hot pressing of hydroxyapatite-magnetite (HA-xFe3O4; x = 0, 5, 10, 20 and 40 wt%) powders in oxidizing and inert atmospheres was carried out and the resulting phases and microstructure were characterized. A detailed analysis of the phase assemblage by Rietveld refinement of the X-ray diffraction (XRD) data and Mössbauer spectroscopy revealed the major retention of Fe3O4 along with wustite (FeO) formation under reducing conditions while hematite (α-Fe2O3) was the oxidized product of conventional sintering in ambient atmosphere. A good correlation between the unit cell volume increases in HA observed from Rietveld refinements and Fe incorporation into the apatite lattice from Mössbauer spectral parameters was evident. Further, the Mössbauer data analysis indicated a preferential occupancy of Fe at the Ca(1) site under oxidizing conditions and Ca(2) site in inert atmosphere. The above phase analyses were further confirmed by X-ray photoelectron spectroscopy (XPS), Infrared spectroscopy (FT-IR) and CHN analysis. The microstructure of the hot-pressed samples observed under transmission electron microscope (TEM) divulged similar phases as deduced from XRD as well as the formation of translational Moire fringe patterns due to inference of overlapping crystal planes of HA and Fe3O4 in the HA-40 wt% composite. Such translational Moire fringes suggest a preferred arrangement and orientation of the crystallites resulting from hot-pressing, which correlated well with the room temperature magnetic measurements made with the help of a vibrating sample magnetometer (VSM). The compositional similarity of Fe doping in HA to that of the tooth enamel and bone presents these HA-Fe3O4 composites as potent dental/ orthopaedic implant materials. In the conclusive study, the hot-pressed HA-xFe3O4 composites were tested for their efficacy in supporting the osteogenesis of human mesenchymal stem cells (hMSCs) assisted by intermittent static magnetic field exposure. The magneto-responsive substrates were applied as platforms for the culture of hMSCs and the effect of static magnetic field (SMF) exposure on the viability, proliferation and differentiation of hMSCs were elucidated. With a mild compromise in viability, SMF triggered the osteogenic differentiation of hMSCs mediated by proliferative arrest in the G0/G1 phase and elevated intracellular calcium levels. The early bone marker genes - Runx2, Col IA and ALP were significantly up regulated upon SMF exposure on pure HA and HA-Fe3O4 composites. Further, the late osteogenic markers – OCN and OPN were detected exclusively in the HA-xFe3O4 (x = 10 and 40 wt%) composites. Matrix mineralization was enhanced and CaP nodules were detected on similar SMF treated HA-Fe3O4 composites. A substrate magnetization and time dependent modulation of gene expression was recorded which corroborated well with the temporal trending of osteogenic genes during bone development. In conclusion, substrate magnetization can be applied as a tool to modulate the behavior of stem cells and direct them towards osteogenic lineage. Such a pertinent combination of substrate magnetization and external magnetic field stimulation can be applied synergistically for stem cell based bone tissue engineering applications.

Indiana University-Purdue University Indianapolis (IUPUI) / Organic-rich substrates such as soil provide an excellent carbon source for bacteria. However, hydrocarbons such as those found in various ignitable liquids can also serve as a source of carbon to support bacterial growth. This is problematic for fire debris analysis as samples may be stored at room temperature for extended periods before they are analyzed due to case backlog. As a result, selective loss of key components due to bacterial metabolism can make identifying and classifying ignitable liquid residues by their chemical composition and boiling point range very difficult. The ultimate goal of this project is to preserve ignitable liquid residues against microbial degradation as efficiently and quickly as possible. Field and laboratory studies were conducted to monitor microbial degradation of gasoline and other ignitable liquids in soil samples. In addition to monitoring degradation in potting soil, as a worst case scenario, the effect of soil type and season were also studied. The effect of microbial action was also compared to the effect of weathering by evaporation (under nitrogen in the laboratory and by the passive headspace analysis of the glass fragments from the incendiary devices in the field studies). All studies showed that microbial degradation resulted in the significant loss of n-alkanes and lesser substituted alkylbenzenes predominantly and quickly, while more highly substituted alkanes and aromatics were not significantly affected. Additionally, the residential soil during the fall season showed the most significant loss of these compounds over the course of 30 days. To combat this problem, a chemical solution is to be immediately applied to the samples as they are collected. Various household and commercial products were tested for their efficacy at low concentrations to eliminate all living bacteria in the soil. Triclosan (2% (w/v) in NaOH) proved to be the most effective at preserving ignitable liquid residues for at least 30 days.