Nanocoatings for preventing orthopaedic implant–associated bacterial infections

The Research department of the Valdoltra Orthopaedic Hospital is determined to conduct more extensive research studies on modification of materials for orthopaedic implants. The starting research in the newly founded Research laboratory shall include basic and applied studies which results shall be potentially considered and eventually implemented in daily clinical practice of Valdoltra Orthopaedic Hospital. With an accurate inspection of the emerging medical needs in the field of orthopaedics we envisaged the urgent need to provide a long-term protection for orthopaedic prostheses. By means of using nano-engineering approaches for the functionalization of orthopaedic implant surfaces with suitable antimicrobial agents, it is possible to protect orthopaedic implants against harmful bacteria, which trigger the initiation of implantassociated bacterial infection. As the implant-associated bacterial infection can affect the longevity of the prosthesis, thus, the scientific and financial efforts, with the help of the project Trans2care, will be focused substantially on the design and fabrication of protective antibacterial coatings for orthopaedic implants

Inhibitory effects of chitosan coating against biofilm formation on metal implants

An effective approach to combat bacteria adhesion onto metallic implants surface is to functionalize the biomaterial surface such that bacterial growth could be impaired or the bacteria are killed upon contact with the surfaces. In the recent years, a majority of the research in material science has been devoted to modification or functionalization of implant surfaces with composite coatings with bactericidal capability such as polymeric coatings. For instance, chitosan (CH) is a polycationic polysaccharide which antibacterial properties and osteoblast function-enhancing nature has received substantial interest. The main goal of our study was to evaluate the effect of different chitosan-coated metals, routinely used in orthopaedics, on the survival of Staphylococcus epidermidis and Staphylococcus aureus. The results clearly showed that survival of attached bacteria onto metals functionalized with chitosan was lower when compared to bacterial survival determined on the surface of unmodified metals. Moreover, chitosan coating caused bacterial cells to lose their regular spherical shape. Thus, the results proved that chitosan could be used as alternative material for the preparation of antimicrobial coatings for implants

Nanostructured mterials for enzyme immobilization.

V doktorski nalogi je nazorno predstavljen postopek sinteze magnetnih nanodelcev iz železovega oksida maghemita, γ-Fe2O3, za vezavo biokatalizatorja. Magnetni nanodelci, ki v zadnjih letih vse bolj pridobivajo na vrednosti kot potencialni encimski nosilci, so bili najprej sintetizirani z metodo obarjanja ali koprecipitacije železovih (II, Fe2+) in železovih (III, Fe3+) ionov v alkalnem mediju pri hitrem mešanju in visoki temperaturi. Površinska funkcionalizacija magnetnih nanodelcev je bila izvedena v dveh stopnjah. Primarna funkcionalna prevleka iz silicijevega dioksida (SiO2), ki delcem daje predvsem višjo stabilnost, je bila sintetizirana pri striktno kontroliranih reakcijskih pogojih iz natrijevega silikata ali Na2SiO3. Sekundarni funkcionalni sloj za doseganje višje funkcionalnosti in reaktivnosti površine nanodelcev je bil sintetiziran iz organskih molekul aminosilana ali 3-(2-aminoetilamino)-propil-dimetoksimetilsilana v kislem mediju. V nadaljnjem so bili tako površinsko spremenjeni magnetni nanodelci uporabljeni za imobilizacijo specifičnega biokatalizatorja holesterol oksidaze (ChOx, EC 1.1.3.6) iz Corynebacterium sp. Analiza magnetnih nanodelcev z imobilizirano ChOx je pokazala, da so imeli nanodelci značilno obliko kroglice ali sfere s povprečnim premerom 50,2 nm. Vezava holesterol oksidaze je bila uspešno potrjena tudi s FT-IR tehniko. Učinkovitost imobilizacije na magnetni nosilec je znašala 92 % pri uporabljeni koncentraciji encima 100 µg mL-1. Aktivnost imobilizirane ChOx na magnetne nanodelce, prevlečene s tanko plastjo silikatne prevleke premera 3 nm, je bila 57 % v primerjavi z aktivnostjo ChOx. Študija vpliva pH-vrednosti in temperature na aktivnost in stabilnost encimskega preparata je pokazala, da ima imobilizirana ChOx višjo toleranco na spremembo pH-vrednosti okolja in višjo termično stabilnost. Prav tako je bila stabilnost imobilizirane ChOx pri ponovni uporabi dobra. V drugem delu doktorske disertacije je opisana priprava aktivnih encimskih skupkov iz encima peroksidaze (HRP, EC 1.11.1.7), pridobljenega iz navadnega hrena (lat.: Armoracia rusticana or Cochlearia armoracia), in postopek zamreženja le-teh z glutaraldehidom za pripravo končne oblike stabilnih zamreženih encimskih skupkov ali na kratko CLEAs. Postopek priprave CLEAs je bil razdeljen na dva ključna dela, in sicer, na obarjanje topnega ali nativnega encima s pomočjo ustreznega obarjalnega reagenta, in na nadaljnjo zamreženje tako izoborjenega encima s pomočjo mrežnega povezovalca. Končni videz CLEAs je podoben motni suspenziji, v kateri so jasno vidni skupki encimov, značilne sferične oblike in premera okrog 250 nm. Uspešno smo sintetizirali zamrežene encimske skupke iz hrenove peroksidaze pod različnimi testnimi pogoji. Končna aktivnost encimskih skupkov iz HRP je bila 83 %. Zamrežene encimske skupke smo sintetizirali v prisotnosti encima albumina iz kokošjih jajc in funkcionalnega aditiva, penta-etilen-heksanamina (PEHA). Encim albumin poveča stabilnost encimskih skupkov, medtem ko PEHA poveča število prostih amino skupin (-NH2) na zunanji površini encima HRP, kar poenostavi postopek zamreženja encimskih delcev z mrežnim povezovalcem.The research work offers a concise guide of the synthesis of maghemite magnetic nanoparticles, γ-Fe2O3, for immobilization of a biocatalyst. Magnetic nanoparticles, that are gaining an exceptionally increased attention as potential enzyme support in the recent years, were synthesized by the coprecipitation technique of ferrous (II, Fe2+) and ferric (III, Fe3+) ions in alkaline medium at harsh stirring and high temperature, respectively. Surface functionalization of magnetic nanoparticles was carried out stepwise and divided into two major steps. Primary functional layer of silica (SiO2) that enhanced the stability of magnetic nanoparticles was synthesized under strictly regulated reaction conditions from sodium silicate. Next, the secondary functional layer formed of organic molecules of amino silane or 3-(2-Aminoethylamino)-propyl-dimethoxymethylsilane in order to achieve higher functionality and reactivity of the surface of magnetic nanoparticles was synthesized in acidic reaction medium. Furthermore, the prepared magnetic nanocomposites were used for the immobilization of a specific biocatalyst cholesterol oxidase (ChOx, EC 1.1.3.6) from Corynebacterium sp. The analysis of the magnetic nanoparticles with immobilized ChOx showed that the nanoparticles adopted a typical spherical shape with a mean diameter of 50,2 nm. The binding of ChOx was successfully confirmed by FT-IR technique. The binding efficiency was 92 % and was maximally achieved at enzyme concentration of 100 µg mL-1. The activity of immobilized ChOx onto magnetic nanoparticles, coated with a thin functional layer of silica with a thickness of 3 nm, was estimated to be 57 % in comparison to its native ChOx. The effects of pH and temperature also indicated the bound ChOx had better pH-tolerance and exhibited higher thermal stability. Furthermore, the immobilized system revealed also good reusable stability. The second part of the PhD work was focused on the synthesis of active enzyme aggregates of peroxidase (HRP, EC 1.11.1.7) from horse radish roots (lat.: Armoracia rusticana or Cochlearia armoracia), further cross-linked with glutaraldehyde in order to obtain the final form of stable cross-linked enzyme aggregates or CLEAs. The procedure to prepare CLEAs was divided into two major steps, that involves first the precipitation of the soluble enzyme with a suitable precipitant such and second the crosslinking step with an appropriate cross-linker. The final suspension of CLEAs obtained was moderately turbid and enzyme particles could be normally observed with an average diameter of 250 nm. The CLEAs of HRP were successfully produced under different testing conditions afterwards. The final recovery activity of the CLEAs attained was 83 % compared to the activity of native enzyme. The synthesised CLEAs were prepared in the presence of egg albumin and functional additive as pentaethylenehexamine (PEHA). The role of albumin in CLEAs preparation plays a significant role as a stabilizing agent of CLEAs particles, whereas the addition of PEHA was fundamental to obtain fully cross-linked HRP aggregates. For, the HRP possesses only 6 Lys (lysine) amino residues, that substantially impedes the cross-linking of the enzyme to completion, the addition of PEHA increased the free amino groups (-NH2) on the outer surface of the enzyme, making the cross-linking more feasible