Común a ambos ejercicios fiscales

Given the severe problems associated with prosthetic infections after total joint replacements, it becomes evident the need to develop antimicrobial bone cements with better performance and outcome for total joint replacement to improve patients’ quality of life.

In this context, the present PhD project aims to develop a novel nano-composite antimicrobial bone cement containing antibiotic or non-antibiotic antimicrob ia l agents or a combination of both, for the prevention of prosthetic infections after total joint replacement which is one of the major causes for revision surgery. The antimicrobial agents to be tested are gentamicin and chlorhexidine, either alone or in combination, to evaluate potential synergism or additive antimicrobial effect needed to overcome the problem of antibiotic resistance mentioned earlier. We hypothesize that the release of antimicrobial agents from the bone cement can be sustained at inhibitory concentrations for a long time (3-6 months) using LbL assembly technique combined with nanotechnology. In order to provide prophylaxis from post-surgical orthopeadic infections, both early and late stage infections. To the best of our knowledge, the LbL assembly technique has not been applied before to control the release of antibiotics from bone cement.

To accomplish this aim, the following objectives were defined:

(i) Novel nanotechnology based antimicrobial drug delivery system development:

In the initial stage, silica nanoparticles were prepared, which are broadly applied as drug delivery carrier because of their biocompatibility, high loading capacity, ease of synthesis and scale up with reasonable cost. Silica nanoparticles were functionalised with amino group to allow further modifications on their surface through the deposition of cationic or anionic polyelectrolytes, and antimicrob ia l agents, during LbL multilayer coating process.

Gentamicin has been employed as a model antibiotic, which is a widely- used aminoglycoside antibiotic in TJR, because of its’ wide spectrum antimicrob ia l activity and thermal stability during the exothermic setting reaction of PMMA bone cement. Gentamicin was loaded on the silica nanoparticles using layer by

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layer assembly (LbL), allowing control of coating thickness and composition at nanoscale level in a reproducible manner. In addition, chlorhexidine has been employed as a model antimicrobial non-antibiotic agent, which is widely used as antiseptic and disinfectants skin infections, cleaning wounds, preventing dental plaque, yeast infections of the mouth, for disinfecting urinary tract catheters and sterilisation of surgical instrument.

Different polyelectrolytes were used to coat the surface of silica nanopartic les during LbL coating process using hydrolysable and non-hydrolysable polymers, controlling the release of gentamicin, chlorhexidine, or a mixture of both from polyelectrolyte multilayers on the surface of silica nanoparticles. In addition, the polyelectrolytes aid in antibiotic loading between different layers by electrostatic attraction between oppositely charged species. Silica nanoparticles with different coatings and loaded antimicrobial agents were characterised by transmission electron microscopy (TEM), thermogravimetric analysis (TGA), zeta potential, Fourier transform infra-red spectroscopy (FTIR), and drug release testing in different release media representing healthy and infected joint.

(ii) PMMA bone cement loaded with the novel antimicrobial nanoparticles: After full characterisation of the newly developed novel LbL multilayer nano-drug delivery system, the nanoparticles with different types of coatings and antimicrobial agents were loaded into the PMMA bone cement. Then, the newly formed nanocomposites were evaluated for the following properties:

1. Bone cement settling time.

The influence of nanoparticles on bone cement settling time was determined through rheological tests, in particular dynamic oscillation tests.

2. Antimicrobial agent release quantification.

the release of antimicrobial agents from bone cement was evaluated and compared against commercial formulations e.g. Cemex G and Palacos R in

vitro.

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The antimicrobial properties were determined once the nanopartic les incorporated into bone cement against common bacteria reported to cause post- orthopaedic surgery infections, such as: Staphylococcus aureus, MRSA, S.

epidermidis and Acinetobacter baumannii. Moreover, they were compared

with the antimicrobial properties of commercial formulations for ALBCs, e.g. Cemex G and Palacos R.

4. Mechanical testing.

Mechanical properties of the nanocomposite were determined against commercial formulations for ALBCs, e.g. Cemex G. In order to assess the effect of incorporated nanoparticles on the mechanical properties of the bone cement, such as compression, bending strength and fracture toughness.

5. Water uptake studies.

When immersed in fluids, the increase of bone cement containing nanoparticles because of water uptake was monitored, and compared to commercial formulations to evaluate the influence of nanopartic les incorporation on the physicochemical properties of the bone cement, in particular hydrophilicity.

6. Nanoparticles distribution:

Fluorescence images were taken for the surface and inside of the bone cement to evaluate the homogeneity of distribution of nanoparticles and compare it to antibiotic powder distribution in commercial cements.

7. Cytotoxicity testing.

The cytocompatibility was verified for the PMMA impregnated bone cement against commercial formulations e.g. Cemex G using relevant cell lines e.g. osteoblasts, to make sure that the newly formed nanocomposite is biocompatible with bone tissue, e.g. Methylthiazolyl tetrazolium (MTT) assay, Lactase dehydrogenase release (LDH) assay test, osteoblast calcium production assay (alizarin red), Nitric oxide production and fluoresce nce imaging.

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2 General methods

This chapter refers to some of the common methods that have been used for the preparation of different nanoparticles and bone cements, and testing their properties throughout this thesis. Later chapters will refer to these methods to reduce repetition with highlighting main differences in nanoparticle and nanocomposite formulation. Test methods that are specific to certain chapters will be described in detail in the appropriate chapter.