In situ Antibiotic Treatment: Eradicating Pathogens at the Site of Infection

Bone infections, or osteomyelitis, are diseases that have existed for many millions of years in biological organisms. Today it is treated by application of surgical interventions and antibiotics, but the number of cases is increasing due to population growth, more comorbidities and more use of bone implants that can become infected. As pathogens are becoming more frequently resistant, new treatment solutions are warranted. In this thesis, the new CarboCell in situ drug delivery technology is investigated for its potential to treat osteomyelitis. CarboCells are organic liquids which comprise sugar esters, organic solvent and co-solvents and can be injected via fine needles to form a depot in an aqueous environment.
Through literature several antibiotics and adjuvants were selected for testing, and a new in vitro biofilm model was developed for the tests. In the model, the most frequent osteomyelitis pathogen, Staphylococcus aureus, was utilized as model organism. Gentamicin, clindamycin, levofloxacin, cis-2-decenoic acid and cis-11-methyl-2-dodecenoic acid were deemed the most promising hits and thus selected as lead compounds. Apart from gentamicin, they were all directly soluble in CarboCell formulations, and their release profiles were tuneable in vitro and in vivo by formulation. They also exhibited an acceptable stability in CarboCells.
The lead compounds were tested in vivo as monotherapy, and in combinations, and proof-of-concept was demonstrated in murine models, in which CarboCell-mediated antimicrobial therapy was capable of prophylactically prevent infection but also eradicate established biofilm infection. Lead compounds were advanced to a well-characterized, translational, implant-associated osteomyelitis porcine model, in which CarboCell-mediated antimicrobial treatment were also capable of infection clearance, without administration of parenteral antibiotics. Previous studies have tested standard-of-care and commercial antibiotic-eluting bone cement treatment alone, but osteomyelitis has never been cleared in the same experimental model, utilizing the same strain of S. aureus.
Gentamicin was not directly compatible with the CarboCell system, but instead was chemically modified by complexation with the hydrophobic compound, docusate. The in vitro and in vivo release of gentamicin·docusate complexes was tuneable by modulating complexation and by CarboCell formulation. The gentamicin·docusate complexes were also potent in vitro, and their in vivo efficacy was tested in rodent and porcine models. All bone- and implant samples tested in the porcine models were cleared of pathogens and no gentamicin resistance was observed in the subcutaneous-residing pathogens that were recovered from four of nine animals. Future studies should investigate whether CarboCellmediated antibiotics confer an added benefit to standard-of-care. 
It was also discovered that when lactose octa-isobutyrate-based CarboCells are formulated with cellulose acetate butyrate and injected in water, the formed depots absorb water, create interconnected pores, and swell, both in vitro and in vivo. This type of CarboCell was termed porous, expanding, biocompatible scaffolds (PEBS). PEBS’ in vivo swelling and pore structures can be monitored by non-invasively by computed tomography if a contrast agent is applied. PEBS were shown to be capable of releasing compounds sustainable over time, and the release profiles were tuneable by formulation. Osteoblast cell lines were shown to be able to attach to PEBS in vitro and proliferate. From qualitative spinning-disc confocal microscopy, PEBS formulation differences gave rise to varying numbers of cell attachment- and proliferative sites. Future studies are needed to elucidate whether PEBS can act as an injectable and osteoconductive void filler in vivo – and if release of bone healing-accelerating agents and antimicrobials will lead to an improved therapeutic outcome in osteomyelitis treatment.