並列タイトル等人工関節周囲感染にかかる細菌株の分光分析および脊椎インプラント用の新抗菌性複合材料
一般注記type:Thesis
A strong demographic demand for arthroplastic devices coupled with a decreased efficacy of antibiotics has been predicted over the next two decades resulting in an exponential increase in the number of periprosthetic joint infections (PJIs). Advanced strategies are therefore required in order to improve the local immune response and suppress the bacterial adhesion and biofilm formation. The use of biomaterials that autonomously counter infections is one possible approach to improve orthopedic outcomes. PJIs result from complex interactions among the host, the pathogen, and the implant. Infections originate from isolated prokaryotic cells which initially adhere to the implant’s surface forming a quorum, followed by the generation of a biofilm which shelters the development of an organized complex bacterial community. The biofilm also protects bacteria from antimicrobial agents. Nowadays, many techniques are used to detect the state and location of these infections cause able to monitor specific bacterial components as DNA, proteins and lipids. One class of these methods is represented by vibrational techniques such as Raman and IR absorption spectroscopy which provide a fast, non-invasive and low-cost analysis, also used for the identification of single bacterial strains. Cellular compounds, functional groups or substructures in the spectrum are represented by specific vibrational bands, making it possible to verify the metabolic changes derived from different processes as growth and aging and from the interaction with pollutants and drugs. The first part of this thesis is focused on the study and comparison of different materials used in orthopedic field and their antibacterial effect against gram-positive S. epidermidis using spectroscopic and microscopic techniques. Firstly, in situ time-lapse Raman spectroscopic experiments were conducted by exposing gram-positive S. epidermidis for 12, 24, and 48 h to silicon nitride (Si3N4) bioceramics and titanium alloy substrates. The goal was to understand the evolution of bacterial metabolism and to elucidate the ceramics antimicrobial behavior focusing the attention on the changes occurred on membrane phospholipids, DNA and protein structures. Results clearly showed the fingerprints of bacterial lysis, as confirmed by conventional fluorescence microscopy probes, suggesting that a localized pH change at Si3N4’s surface induced variations in peptidoglycan layer leading to membrane degradation and cellular death. This pH modification brought an increase of amine groups which improved the efficiency of the substrates. Subsequently we analyzed by means of Raman spectroscopy and Fourier transform infrared spectroscopy (FTIR), Si3N4 and zirconia-toughened alumina (ZTA) after different time of S. epidermidis exposure. The purposes were: (i) to compare the antibacterial performance of Al2O3-based (oxide) and Si3N4 (non-oxide) ceramics, for the first time, in the same series of biological tests, (ii) to quantitatively assess their respective abilities to inhibit biofilm formation, and (iii) to propose mechanisms for their observed bacteriostatic behaviors. In combination with statistical validation, pH measurements and other biological tests, it’s been possible to describe how, unlike ZTA, Si3N4 possesses an inherently anti-infective surface chemistry, which acts in a responsive way against bacterial loading elucidating the mechanism detailed. The second part of this thesis is focused on the in situ vibrational analyses of the metabolic response of gram-negative Escherichia coli after exposure to the three different type of substrates (Si3N4, ZTA and Titanium Grade 5 alloy). The metabolic pathways, before and after bacterial exposure, were monitored by means of Raman and FTIR spectroscopies and fluorescence microscope analysis. The results indicated a constant decrease of cellular compounds markers concentration associated to the development of significant osmotic stress in the bacterial strain over time upon exposure to Si3N4 comparing with the other samples indicating how the antibacterial behavior exerted by non-oxide bioceramic was more effective than that observed on biomedical titanium alloy. The metabolic rates changed rapidly, the bacterial membrane was damaged, and complete lysis occurred within 48 h exposure. Conversely, on ZTA bioceramic oxide substrate bacteria proliferation and no lysis was observed.In the last part of this work, we investigated the quality of a new developed composite based on a PEEK matrix where fractions of Si3N4 have been incorporated. PEEK’s has poor antibacterial resistance lacks bioactive effects, and is transparent in radiographies. Combining this material with silicon nitride, the goal was to balance these deficiencies while preserving the base polymer’s biocompatibility, chemical stability, and also the low elastic modulus. Using three Si3N4 variants, β-Si3N4, α-Si3N4, and β-SiYAlON the composite tested in vitro showed bacteriostatic properties versus S. epidermidis bacteria and an improvement in the polymer’s osteoconductivity versus SaOS-2 line cells comparing with the untreated PEEK. This simple fabrication method which exploits Si/N chemistry could be used to produce new materials in order to beneficially replace monolithic PEEK implants.
連携機関・データベース国立情報学研究所 : 学術機関リポジトリデータベース（IRDB）（機関リポジトリ）