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This work, comprising four studies, focuses on experimental implants and materials for orthopaedic applications designed to address the often concomitant issues of infections and bone loss. The first two studies examine silver (Ag) as an antibacterial agent and its effects on human cells when used as a coating on titanium (Ti) implants. The last two studies explore methods for regenerating bone defects.

In the first study, we compared two types of Ag-coated Ti implants provided by an implant manufacturer. One implant is used clinically, while the other was experimental and contained smaller amounts of Ag. Physicochemical analysis showed that the entire surface of the clinical implant was coated with Ag, while the Ag on the experimental implant formed aggregates on the surface. The clinically used implant released significantly more Ag, while, the experimental implant's release ceased after a few days. S. aureus and osteoblasts was cultured separately then on the implants, and we showed that smaller amounts of Ag on the implants maintained satisfactory antibacterial effects while minimising adverse effects on the osteoblasts.

The second study investigated the effects of different ionic Ag concentrations on osteoblasts and mesenchymal stem cells. Using various methods, including PCR, enzymatic analyses for cell differentiation, microscopy, and staining for mineralisation, we found that even small amounts of Ag could inhibit mineralisation by human osteoblasts. We observed no significant impact on the osteogenic differentiation of osteoblasts or mesenchymal stem cells regarding gene expression and ALP production. However, microscopic analysis revealed abnormal cell patterns, such as reduced confluence in Ag-treated groups compared to controls. These findings, suggest that Ag-coated implants should be used cautiously in clinical settings, especially in parts of the prosthesis intended for direct bone integration.

Study III employed the freeform reversible embedding of suspended hydrogels (FRESH) technique, to 3D-print collagen structures with trabecular geometry. This technique and its structures were compared to stereolithographic printing, an alternative 3D printing method, which produced analogous structures using a different material, polyethylene glycol diacrylate (PEGDA). Rheological and mechanical analyses were performed on the 3D-printed collagen structures to characterise their elasticity and stiffness; electron microscopy was used to map the surface geometry of the collagen and PEGDA structures. Cultures of human osteoblasts and osteosarcoma cell lines on the two types of structures showed that the 3D-printed collagen structures were better suited as carriers for osteogenic cells compared to the 3D-printed PEGDA structures.

In the fourth study, we successfully 3D-printed artificial bone of collagen and hydroxyapatite. The collagen in this material was modified by cross-linking. Mechanical and rheological analyses of the material in gel and solid forms were performed, and the distribution pattern of hydroxyapatite within the material was examined. Finally, we cultured human osteoblasts on the 3D-printed collagen-hydroxyapatite structures up to four weeks. We found that osteoblasts grew well and differentiated satisfactorily on structures printed with our new material.

Overall, this work shows how surface coatings and 3D printing might solve the two biggest issues in orthopaedics—infections and missing bone. We trust this dissertation will illuminate the underlying scientific principles of these techniques for orthopaedic surgeons.

Tissue engineering aims to develop materials that help the body repair damaged tissues. Collagen is attractive for this purpose because it is a major component of the extracellular matrix, but conventional collagen hydrogels are mechanically weak, while dense collagen is more difficult to process and shape. This thesis investigated how collagen could be engineered into stable, printable and biologically accessible scaffolds.

Study I examined three-dimensional printing of conventional collagen using a support-bath technique. The method enabled controlled scaffold architecture, but the resulting collagen remained mechanically soft. The study showed that geometry could be imposed on collagen, while also revealing the need for greater structural function within the material itself.

Study II therefore focused on developing a dense collagen–silica hybrid, DCSi. A concentrated acidic collagen precursor containing hydrolyzed silanes remained processable before fabrication and was subsequently matured in alkaline borate. The maturation environment strongly influenced collagen organization, silica distribution and mechanical behavior. Borate produced a dense fibrillar collagen structure with finely distributed silica and substantially improved structural stability. DCSi could also be printed into multilayer structures when extrusion and stabilization were controlled together. Importantly, cells colonized the material in vitro, while host cells and blood vessels entered the scaffold after implantation.

Study III adapted DCSi for bone repair by introducing a calcium-phosphate mineral phase. Mineralized DCSi retained its collagen-rich structure and flexibility while providing a bone-specific inorganic component. After implantation in a rat femoral defect, mineralized DCSi produced more new bone than non-mineralized DCSi after six weeks.

Study IV examined DCSi as a printed patch for uterine tissue repair. The patches were designed as thin and flexible structures and were evaluated with or without mesenchymal stromal cells. Both supported tissue ingrowth and vascularization, while the cell-free patches showed more consistent structural regeneration under the conditions tested.

Together, these studies show that dense collagen can be engineered into a versatile scaffold platform by controlling how structure develops during processing. This provides a route towards collagen-based materials that are not only biologically relevant, but also structurally functional, manufacturable and adaptable to different regenerative applications.

The cellular microenvironment provides biochemical and biomechanical cues that regulate cell behavior. Developing in vitro models that capture selected features of these complex environments enables the investigation of cell responses in healthy and pathological conditions. The overall aim of this thesis was to employ biomaterials, additive manufacturing and engineered tools to integrate microenvironmental features into studies of cell responses.

In Study I, we developed an active-mixing bioprinting toolhead to automate the extrusion of hydrogel compositional gradient arrays onto cell monolayers. We demonstrated the tool’s performance with alginate hydrogels and highly concentrated engineered spidroins, and confirmed its compatibility with screening cell responses in multi-well plates. Cancer cell sensitivity to an apoptosis-inducing drug increased along a gradient array of increasing extracellular laminin concentrations, demonstrating the potential of this approach to evaluate the influence of specific microenvironmental cues on cell responses.

In Study II, we modified the toolhead to generate cell-laden hydrogel arrays with integrated drug gradients. We evaluated hydrogel mixing and cell viability under different operational parameters, and applied the system to screen responses of hydrogel-embedded cancer cells to gradients of the ferroptosis-inducing drug erastin. This proof-of-concept study establishes the feasibility of this approach for screening cell responses in three-dimensional microenvironments.

In Study III, we applied embedded bioprinting to fabricate millimetre-scale bone-like collagen scaffolds with trabecular structures, and evaluated their ability to support cells. Although the scaffolds exhibited limited mechanical strength, they supported osteoblastic cultures expressing osteogenic markers, highlighting the potential of embedded printing to generate osteoconductive collagen scaffolds mimicking trabecular bone architecture.

In Study IV, we applied our cell-compression platform to evaluate the capacity of biomaterial scaffolds, developed for load-bearing implants, to transmit mechanical forces to cells. Melt-electrowritten porous poly(ε-caprolactone) scaffolds were used to compress pre-osteoblast cell monolayers while changes in cytosolic calcium were imaged. Mechanoresponses differed according to the local scaffold architecture, demonstrating the platform’s potential for in vitro assessment of scaffolds intended to interface with dynamic microenvironments in vivo.

Together, these bioengineered approaches enabled the investigation of cell responses within controlled microenvironments incorporating selected aspects of the complexity encountered in vivo.