The Development of Antibiotic-Free 3D Printed Composite Scaffolds for the Prevention of Infected Bone Defects
Creators: name: Genoud, Katelyn
Publisher: name: Royal College of Surgeons in Ireland
Year: 2024
Subjects: classificationCode: 400302
subject: Biomaterials
classificationCode: 400303
subject: Biomechanical engineering
classificationCode: 400399
subject: Biomedical engineering not elsewhere classified

description: Severe bone infection, known as osteomyelitis, is predominately caused by Staphylococcus aureus (S. aureus) bacteria and typically necessitates long term antibiotics as well as full surgical debridement of the infected tissue, which can compromise the self-regenerative capacity of bone leaving behind a large ‘dead space’ requiring bone grafting. Therefore, the development of a multifunctional non-antibiotic antimicrobial biomaterial that could locally fight bacteria while providing a template for osseous tissue ingrowth would be a major development. Thus, the overall aim of the research presented in this thesis was to develop a non-antibiotic antimicrobial collagen-based scaffold for the prevention of osteomyelitis that has the potential to be used in load-bearing indications. 
In the study presented in Chapter 2 of this thesis, 3D printed frameworks were developed and used to reinforce collagen-nanohydroxyapatite scaffolds to enhance their mechanical properties without altering the highly porous microarchitecture of the matrix which supported cellular infiltration, proliferation, and osteogenic differentiation. The results presented thus demonstrate the potential of 3D printing as an enabling technology to enhance the mechanical properties of tissue engineered scaffolds for different applications, specifically in load-bearing applications and larger defect sites following injury or infection. 
In Chapter 3, the dose of silver doped within hydroxyapatite, and the concentration of silver-doped hydroxyapatite (AgHA) within collagen scaffolds were optimised to achieve both pro-osteogenic and antibacterial properties. Specifically, the scaffolds with formulations of 0.75-200% collagen-AgHA and 1.5-200% collagen-AgHA demonstrated osteogenic capacity to the same extent as pristine hydroxyapatite(0-200% collagen-AgHA)while also exhibiting potent antibacterial activity against S. aureus, the primary causative pathogen of osteomyelitis. 
In the study presented in Chapter 4 of this thesis, collagen-AgHA scaffolds, developed in Chapter 3, with optimisedosteogenic and antibacterial properties were reinforced using a 3D printed framework, from Chapter 2, to achieve enhanced mechanical properties to expand its use for load-bearing situations. The reinforced collagen-AgHA scaffold supported cellular infiltration and osteogenic differentiation of clinically relevant primary bone marrow-derived mesenchymal stem cells in vitro, and the addition of the 3D printed framework did not alter the antimicrobial properties of the scaffold. Overall, a distinct and novel implant was developed with expanded use for load-bearing applications for the prevention of infected bone defects.
In Chapter 5, a femoral defect model using female rats was successfully refined which allowed for the subsequent in vivo evaluation of the regenerative potential of the reinforced collagen-AgHA and reinforced collagen-hydroxyapatite scaffolds. Most importantly, the reinforced collagen-AgHA scaffolds, with proven in vitroantibacterial properties, successfully repaired bone defects in vivo and supported enhanced calcified tissue formation and enhanced vessel infiltration to a larger extent than the non-reinforced scaffolds. 
In Chapter 6,an alternative method to produce the implant was investigated and thus novel 3D printable bioinks were fabricated using polycaprolactone and AgHA. Using the bioinks, a 3D printed scaffold was developed and fabricated with requisite properties for bone tissue engineering, including a stiffness within the range of trabecular bone and optimalporosity to support cellular infiltration and proliferation. The scaffolds successfully supported cellular attachment and osteogenic differentiation, in addition to potent antibacterial properties towards S. aureus. Overall, a novel single step fabrication process was developed to successfully fabricate 3D printable, antibacterial, and osteogenic bioinks and a novel 3D printed scaffold was designed and demonstrated significant potential to be used as an implant for the prevention of infected bone defects. 
Collectively, the research carried out in this thesis has led to the development of antibiotic-free 3D printed composite scaffolds for the treatment of infected bone defects. Through the combination of 3D printing, collagen-based scaffolds with tailored osteogenic properties, and incorporation of metal-based antimicrobials, novel implants for the prevention of large, weight-bearing infected bone defects were successfully fabricated and demonstrated antibacterial and osteogenic properties in vitro as well as pro-osteogenic and angiogenic properties in vivo. In summary this thesis presents potential off-the-shelf  or patient specific preventative implants for osteomyelitis which might reduce the need for antibiotics and bone grafting, thus reducing economic burden and enhancing patient quality of life.