Hydroxyapatite (HA) has been widely used as a bone substitute and more recently as a carrier for local delivery of bone targeted drugs. Majority of the approved HA based biomaterials and drug carriers comprise of micrometer sized particulate HA (mHA) or granules and can therefore only be used for extracellular drug release. This shortcoming could be overcome with the use of cell penetrating HA nanoparticles (nHA) but a major concern with the clinical use of nHA is the lack of data on its in vivo biodistribution after implantation. In this study, we aimed to study the in vivo biodistribution of locally implanted nHA in a clinically relevant tibial void in rats and compare it with mHA or a combination of mHA and nHA. To enable in vivo tracking, HA particles were first labelled with 14C-zoledronic acid (14C-ZA), known to have a high binding affinity to HA. The labelled particles were then implanted in the animals and the radioactivity in the proximal tibia and vital organs was detected at various time points (Day 1, 7 and 28) post-implantation using scintillation counting. The local distribution of the particles in the bone was studied with micro-CT. We found that majority (>99.9%) of the implanted HA particles, irrespective of the size, stayed locally at the implantation site even after 28 days and the findings were confirmed using micro-CT. Less than 0.1% radioactivity was observed in the kidney and the spleen at later time points of day 7 and 28. No pathological changes in any of the vital organs could be observed histologically. This is the first longitudinal in vivo HA biodistribution study showing that the local implantation of nHA particles in bone is safe and that nHA could potentially be used for localized drug delivery.

Treatment of chronic osteomyelitis by radical debridement and filling of the dead space with antibiotic containing calcium sulfate/hydroxyapatite (CaS/HA) bone substitute has shown excellent long‐term outcomes. However, in extensive infections, sessile bacteria may remain in bone cells or soft tissues protected by biofilm leading to recurrences. The primary aim of this study was to evaluate if systemically administrated tetracycline (TET) could bind to pre‐implanted HA particles and impart an antibacterial effect locally. In vitro studies indicated that the binding of TET to nano‐ and micro‐sized HA particles was rapid and plateaued already at 1 h. Since protein passivation of HA after in‐vivo implantation could affect HA–TET interaction, we investigated the effect of serum exposure on HA–TET binding in an antibacterial assay. Although, serum exposure reduced the zone of inhibition (ZOI) of Staphylococcus aureus, a significant ZOI could still be observed after pre‐incubation of HA with serum. We could in addition show that zoledronic acid (ZA) competes for the same binding sites as TET and that exposure to high doses of ZA led to reduced TET–HA binding. In an in‐vivo setting, we then confirmed that systemically administered TET seeks HA particles that were pre‐implanted in muscle and subcutaneous pouches in rats and mice respectively, preventing HA particles from being colonized by S. aureus. Clinical Significance: This study describes a new drug delivery method that could prevent bacterial colonization of a HA biomaterial and reduce recurrences in bone infection.

Abstract. Introduction: biomaterials combined with antibiotics are routinely used for the management of bone infections. After eluting high concentrations of antibiotics during the first week, sub-inhibitory concentrations of antibiotics may lead to late repopulation of recalcitrant bacteria. Recent studies have shown that systemically given antibiotics like tetracycline and rifampicin (RIF) could seek and bind to locally implanted hydroxyapatite (HA). The aim of this in vivo study was to test if systemically administered rifampicin could replenish HA-based biomaterials with or without prior antibiotic loading to protect the material from late bacterial repopulation. Methods: in vivo accretion of systemically administered RIF to three different types of HA-based materials was tested. In group 1, nano (n)- and micro (m)-sized HA particles were used, while group 2 consisted of a calcium sulfate/hydroxyapatite (CaS/HA) biomaterial without preloaded antibiotics gentamycin (GEN) or vancomycin (VAN), and in group 3, the CaS/HA material contained GEN (CaS/HA+GEN) or VAN (CaS/HA+VAN). The above materials were implanted in an abdominal muscle pouch model in rats, and at 7 d post-surgery, the animals were assigned to a control group (i.e., no systemic antibiotic) and a test group (i.e., animals receiving one single intraperitoneal injection of RIF each day (4 mg per rat) for 3 consecutive days). Twenty-four hours after the third injection, the animals were sacrificed and the implanted pellets were retrieved and tested against Staphylococcus aureus ATCC 25923 in an agar diffusion assay. After overnight incubation, the zone of inhibition (ZOI) around the pellets were measured. Results: in the control group, 2/6 CaS/HA+GEN pellets had a ZOI, while all other harvested pellets had no ZOI. No pellets from animals in test group 1 had a ZOI. In test group 2, 10/10 CaS/HA pellets showed a ZOI. In test group 3, 5/6 CaS/HA+GEN and 4/6 CaS/HA+VAN pellets showed a ZOI. Conclusions: in this proof-of-concept study, we have shown that a locally implanted biphasic CaS/HA carrier after 1 week can be loaded by systemic RIF administration and exert an antibacterial effect. Further in vivo infection models are necessary to validate our findings.

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