Developing ex vivo models for understanding microbial infection associated with hip and knee implants

Khan, Jabur 2022. Developing ex vivo models for understanding microbial infection associated with hip and knee implants. PhD Thesis, Cardiff University. Item availability restricted.

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Abstract

Bacterial infections surrounding orthopaedic implants hinder the process of bone repair and prevent osteointegration leading to surrounding bone loss and subsequent implant loosening post-loading. However, the relationship between a commonly occurring implant infectious organism, Staphylococcus aureus (S. aureus) and the local bone marrow cell population is poorly understood. No adequate ex vivo model to investigate the mechanism leading to bacterial-induced osteolysis currently exists. This research sought to develop and characterise a novel, ex vivo bone marrow co-culture model to investigate specific cell responses following S. aureus infection. Slices of rat femurs were cultured up to 48 h either submerged within the culture media (base-type culture) or suspended at a liquid/air interface (Trowell-type culture). Both culture models demonstrated significant cell loss and cell death, however, the Trowell-type culture had better maintenance of cell numbers. Cellular analysis indicated the reduced cell numbers of monocytes, macrophages and neutrophil populations over 48 h. Supplementation of the Trowell-type culture with macrophage-colony stimulating factor (M-CSF) and receptor activator of the nuclear factor kappa B ligand (RANKL) to the culture media maintained CD14 and CD68 positive monocyte and macrophage populations but showed no significance when compared to slices without M-CSF and RANKL. The addition of the cytokines at 20 ng/mL M-CSF and 30 ng/mL RANKL maintained low numbers of multinucleated CD68 osteoclast-like cells at 24 h and 48 h. This research further established that the ex vivo model could be manipulated by microinjection of cytokines at a concentration of 20 ng/mL M-CSF and 30 ng/mL RANKL with or without tibial bone marrow cells into the bone marrow cavity with improved cell numbers compared to non-injected slices. Microinjection of cytokines into the bone marrow cavity with and without tibial bone marrow cells had reduced extracellular LDH release along with improved CD14 and CD68 cell numbers over 48 h in culture. Following infection with either S. aureus NCTC 6571 or the NCTC 7791 strains, cells responded with significant loss of cells along with increased necrosis and apoptosis. Infected slices demonstrated an increase of pro-inflammatory cytokines Tumor Necrosis Factor alpha, interleukin (IL)-1β, and IL-6. The anti-inflammatory levels following infection were low for both IL-4 and IL-10. Histomorphology assessment identified S. aureus colonies within the bone marrow cavity and in the case of the NCTC 7791 type strain, there was the presence of a biofilm-like structure attached to the surrounding cortical bone. This research demonstrates the challenges in developing an ex vivo bone marrow culture model which can support immune cell populations which play key roles in bone resorption. Post-infection, there was an increased presence of pro-inflammatory cytokines and increased cellular necrosis and apoptosis. The ex vivo bone marrow microenvironment can be manipulated with cells, supplements and bacteria. The model, therefore, has the potential to observe interactions of specific cells to the presence of bacterial species within a three-dimensional model. With all results considered, this model has the potential to further understand the processes involved during early orthopaedic infections whilst aiding in the development and assessment of novel therapeutic interventions.

Item Type:	Thesis (PhD)
Date Type:	Completion
Status:	Unpublished
Schools:	Dentistry
Subjects:	R Medicine > RM Therapeutics. Pharmacology R Medicine > RS Pharmacy and materia medica
Uncontrolled Keywords:	Bone models; Ex vivo; Infection model; Staphylococcus aureus;3D tissue model; Bone marrow infection Orthopaedic infections; Hip infections; Knee infections; Prosthetic joint infections
Date of First Compliant Deposit:	28 January 2022
Last Modified:	11 Mar 2023 02:28
URI:	https://orca.cardiff.ac.uk/id/eprint/147020

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