Van Staden, Anton Du Preez
ENGLISH ABSTRACT: Infection is one of the major causes of increased morbidity and the escalating costs associated with orthopedic surgery. The areas that are infected are often difficult to reach and thus difficult to treat. In some surgeries antibiotic-loaded bone cements are used to control infection. Polymethylmethacrylate (PMMA) and calcium phosphate-based bone cements (CPC) are usually used as bone fillers. CPC are bioresorbable and biocompatible (unlike PMMA cements), but can only be used in non- or low-load bearing areas and are thus more applicable in cranio-and maxilla-facial surgeries. Several in vitro and in vivo trials have been conducted on the incorporation of antibiotics and other therapeutic agents into CPC and the release of these agents. As with any solid matrix, release is defined by specific parameters, i.e. matrix porosity, solubility of the drug and interaction of the drug with the cement.
The increase in antibiotic-resistant pathogens, mainly as a result of overuse of antibiotics, has a major impact on the choice of antibiotics that are used in the treatment of bacterial infections. The search for alternative antimicrobial compounds that are active against resistant pathogens, is thus of utmost importance.
Antimicrobial peptides (bacteriocins) produced by lactic acid bacteria may pose a possible alternative to antibiotics. Some of these peptides are active against antibiotic-resistant pathogens. Bacteriocins are small cationic, hydrophobic, or amphiphilic peptides active against a narrow range of target organisms. Most of these peptides are active in the nanomolar range. It may then be advantageous to incorporate bacteriocins into CPC to evaluate if they may be used as an alternative to antibiotics.
The aim of the project was to evaluate if bacteriocins could be successfully incorporated into self seting brushite bone cement and remain effective in vivo without altering basic cement characteristics. Incorporation of bacteriocins into CPC is a novel concept. The low setting temperature and pH of CPC renders it the ideal matrix for incorporation of antimicrobial peptides. In this study, peptide ST4SA, a class IIa broad-spectrum bacteriocin, has been incorporated into brushite bone cement and characterized in vitro. Incorporation of the peptide did not have a significant effect on the crystal entanglement or setting reaction of the cement. Peptide ST4SA was rapidly released and inhibited the growth of the target strain effectively. In another experiment, peptide ST4SA was suspended in poly (lactide-co-glycolide) and electrosprayed to form micro particles that were entrapped in brushite cement. Association of the peptide with microparticles resulted in a delayed release from the cement, followed by a constant release. Nisin F, a class Ia bacteriocin was also incorporated into brushite cement and its activity studied in vitro and in vivo. Similar results were observed in vitro as recorded with peptide ST4SA incorporated into brushite cement. Small cylinders of brushite cement loaded with nisin F were implanted into subcutaneous pockets in mice and each pocket infected with a bioluminescent strain of Staphylococcus aureus (Xen 36). Nisin F in the bone cement prevented the growth of S. aureus in the wound and controlled infection.
With this study we have shown that antimicrobial peptides that differ in structure (classes I and II) could be incorporated into bone cement and control the growth of S. aureus in vivo and in vitro. The mode of action of these peptides differs from antibiotics in that they form a permanent pore in the cell membrane of the target organism. This minimizes the chance of a strain becoming resistant to the peptide. Incorporation of antimicrobial peptides into bone cement may be a possible alternative to antibiotics in the control of bacterial infections associated with implants. AFRIKAANSE OPSOMMING: Infeksie is een van die grootste bydraende faktore tot sterftes en verhoogde kostes in ortopediese chirurgie. Geinfekteerde areas is dikwels moeilik bereikbaar en dus ook moeilik om te behandel. In sommige operasies word antibiotika-gelaaide beensement gebruik om infeksie te beheer. Polymetielmetakrilaat (PMMS) en kalsium fosfaat gebaseerde beensement (KFS) word gebruik as been vullers. KFS is bioverenigbaar en bio-absorberend (in teenstelling met PMMS), maar kan slegs in geen- of liggewig-draende areas gebruik word en is dus van groter toepassing in skedel-, kaak- gesig- en mondchirurgie. Verskeie in vitro en in vivo toetse is al gedoen op die inkorporering van antibiotika en ander terapeutiese middels in KFS en die vrystelling daarvan uit die matriks. Soos met enige soliede matriks is vrylating van die geinkorporeerde bestanddeel afhanklik van sekere parameters, onder andere porositeit, oplosbaarheid van die middel, en die interaksie van die middel met beensement.
Die toename in antibiotika-weerstandbiedende patogene plaas geweldige druk op die keuse van antibiotika wat gebruik word in die beheer van bakteriese infeksie. Die soeke na alternatiewe antimikrobiese middels aktief teen bestande patogene is dus van kardinale belang.
Antimikrobiese peptiede (bakteriosiene) gepproduseer deur melksuur bakteriee mag dalk . alternatief tot antibiotika wees. Sommige van hierdie peptiede is aktief teen verskeie weerstandbiedende patogene. Bakteriosiene is kationiese, hidrofobiese of amfifiliese peptiede wat naverwante bakteriee inhibeer of doodmaak. Die meeste van hierdie peptiede is aktief op nanoskaal vlak. Dit mag dalk dus voordelig wees om bakteriosiene in been sement te evalueer as moontlike alternatiewe tot antibiotika.
Die doel van die proejek was om te evaleer of bakteriosiene suksesfol in "brushite" sement geïnkorporeer kan word en steeds effektief in vivo bly sonder om die basiese eienskappe van die sement te verander. Inkorporasie van bakteriosiene in KFS is 'n nuwe konsep. Die lae stollingstemperatuur en pH van KFS maak dit moontlik om bakteriosiene daarin te inkorporeer. In hierdie studie is peptied ST4SA, . klas IIa wye-spektrum bakteriosien, in "brushite" sement geïnkorporeer en in vitro bestudeer. Die toevoeging van die peptied het nie 'n beduidende effek op die stolreaksie of kristal verstrikking van die sement gehad nie. Peptied ST4SA is effektief vrygelaat en het die groei van die teikenorganisme suksesvol onderdruk. In 'n ander eksperiment is peptied ST4SA in poli (D,L-laktied-ko-glikolied) gesuspendeer en met behulp van elektrosproeiing tot mikropartikels omvorm en is in "brushite" sement geïnkorporeer. Assosiasie van die peptied met mikropartikels het die inisiële vrylating van die peptied vertraag, gevolg deur 'n konstante vrylating. Nisien F, . klas Ia lantibiotikum, is ook in "brushite" sement geïnkorporeer en die aktiwiteit daarvan in vitro en in vivo bestudeer. Die in vitro eienskappe is soortgelyk aan die eienskappe wat vir peptied ST4SA-gelaaide sement waargeneem is. Klein stafies "brushite" sement, waarin nisien F geïnkoproreer is, is in onderhuidse sakkies in muise geplaas en die area met 'n bio-liggewende bakterie (S. aureus Xen 36) geïnfekteer. Nisien F in die beensement het die groei van S. aureus in die wond onderdruk en infeksie beheer.
Met hierdie studie het ons bewys dat bakteriosiene wat struktureel van mekaar verskil (klasse I en II) in beensement geïnkorporeer kan word en die groei van S. aureus in vitro en in vivo kon beheer. Die wyse waarop hierdie peptiede die groei van sensitiewe organismes inhibeer verskil van die van antibiotika deurdat dit porieë in die selmembraan vorm. Die moontlikheid dat organismes weerstandbiedend raak tot die peptied is dus heelwat skraler. Die insluit van antimikrobiese peptiede in beensement mag dalk 'n alternatief tot antibiotika wees in die voorkoming van bakteriële infeksie geassosieer met ortopediese chirurgie. Description:
Masters Degrees (Microbiology)