Selected Publications
January 2023
Systemic rifampicin shows accretion to locally implanted hydroxyapatite particles in a rat abdominal muscle pouch model
Journal of Bone and Joint Infection
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+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, +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+GEN and +VAN pellets showed a ZOI. Conclusions: in this proof-of-concept study, we have shown that a locally implanted biphasic CaS/
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+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, +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+GEN and +VAN pellets showed a ZOI. Conclusions: in this proof-of-concept study, we have shown that a locally implanted biphasic CaS/
Aim There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN). Methods The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites. Results Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF. Conclusion Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections. Osteosarcoma is a malignant cancer of the bone mainly affecting adolescents. Despite progress, the clinical management of osteosarcoma is still challenging. With the current chemotherapy protocol being used for more than 30 years, the number of poor responders is increasing. Although new treatments have been explored since then, no improved tumor eradication effect have been found. In the present thesis, we have developed a new treatment method for osteosarcoma, using hydroxyapatite (HA) based materials as a platform for local delivery of cytostatics. Doxorubicin (DOX), a cornerstone osteosarcoma drug, was chosen as a drug candidate, due to its binding capacity to HA. Different types of HA-based biomaterials were tested for local or targeted delivery of DOX. The efficacy of the developed system was evaluated in-vitro, in osteosarcoma cells as well as in-vivo, in mice bearing an aggressive osteosarcoma.
December 2022
December 2022
December 2022
In Study 1, a clinically approved calcium sulphate (CaS)/HA biomaterial achieved a sustained and controlled release of DOX up to 28 days, both in-vitro and in-vivo. Compared to no treatment or the clinical standard with systemic DOX administration, the local delivery of DOX using a CaS/HA biomaterial significantly hindered tumor progression by inhibiting angiogenesis and cell proliferation.
In Study 2, we investigated the physicochemical interactions between DOX and different sizes of HA particles, both in-vitro and in-vivo. When delivered by HA nanoparticles, DOX is routed to the mitochondria causing insufficient ATP synthesis, less cell migration and cell apoptosis. This leads to stronger in-vivo tumor eradication compared to systemic administration of DOX. Furthermore, nHA mediated delivery of DOX may prevent further metastases in- vivo, which was indirectly verified by PET/CT data.
In Study 3, HA particles (nHA, mHA or n/mHA) were labelled with carbon 14 (14C) to detect particle migration in- vivo. During the observational time of 28 days, the majority (>99.9%) of implanted HA particles, irrespective of the size, stayed in the implantation site (proximal tibia), without migrating to other vital organs. No pathological changes were detected in the vital organs.
In summary, we describe a new and efficient method to supplement osteosarcoma treatment, with a possible rapid translational potential, using clinically approved constituents. By using a hydroxyapatite-based biomaterial, DOX could be routed to the tumor site, more efficiently and with less side effects compared to systemic administration. The chemical interaction between DOX and HA lead to a sustained and controlled DOX release which further improved its tumor eradication effect. When using HA nanoparticles, DOX could be directed to the mitochondria causing tumor cell starvation, reduced migration and apoptosis, jointly leading to improved tumor eradication. The local administration of HA particles, irrespective of size, was confirmed as safe without damage to vital organs. In the future, chemotherapeutics with multi-release profile potentially could be applied by using a combination of nHA and mHA.