• bioengineering
• bioprinting
• bone tissue engineering
• in vitro model
• osteoporosis

Osteoporosis is a worldwide disease caused by an imbalance between bone remodelling carried by osteoblasts and bone resorption by osteoclasts. This is characterized by a reduction in bone mass and an alteration of bone architecture, resulting in bone fragility and increased risk of fractures. Current treatments are traditionally based on surgical procedures, with fixation mechanisms being the gold standard for repairing bone fractures. However, due to reduced bone mass and strength, these mechanisms fail to properly reunion bone fragments. Besides, to counteract the loss of bone mass, patients are usually treated with anti-osteoporotic drugs, which may cause side effects and other complications.
In this context, Chapter 1 presents the advances in bioengineering that can offer promising solutions to manage bone diseases such as osteoporosis. In the treatment of osteoporotic fractures, many researchers have been studying novel approaches to manage the high fracture risk in osteoporotic patients. Regarding long bone fractures, a different strategy is presented in Chapter 2, where in the framework of the European-funded GIOTTO project (grant agreement No 814410), a drug-device combination product was developed, specifically to promote the healing of periprosthetic fractures in osteoporotic patients. The device is 3D printed with a polyester-based blend filament and its surface is functionalised with a drug molecule, ICOS-Fc, which can stimulate bone regeneration and wound healing by inhibiting osteoclast activity. This is placed at the target site using existing fixation mechanisms (e.g., metal plates, screws…) that also contribute to the fracture stabilisation. As a support for the device design, the PhD project initially focused on implementing a in silico model to predict the degradation of the drug-device and thus the release of its by-products containing the drug molecule. Based on a mathematical model for hydrolytic degradation, the model was applied to different geometries to evaluate the release and diffusion of the degradation products in space and time, with the aim to obtain a controlled degradation behaviour. In Chapter 4, the results of the model were used as inputs for the design of a drug-device prototype for a preclinical study on large animals, which was fabricated using a novel 3D bioprinting platform presented in Chapter 3.
Additive Manufacturing technologies have been widely used in the fabrication of implantable bone devices both in the orthopaedic and tissue engineering fields. A recent trend is the combination of different techniques and materials to create multimaterial 3D structures, thus overcoming the limitations of using a single technique/material. In this context, Chapter 3 presents a new custom-designed multimaterial 3D bioprinting platform (named as BOOST), which was upgraded during the PhD with new bioprinting modules for Fused Deposition Modelling (FDM) and thermal Drop-On-Demand (DOD) inkjet printing (IJP). The functionality of the BOOST 3D bioprinter was validated by demonstrating the ability to fabricate multimaterial scaffolds combining FDM and IJP. First, a proof-of-concept was presented to demonstrate their simultaneous use in the same printing process, which consisted of alternating FDM printing of a thermoplastic filament and IJP of black ink. Furthermore, a case-study demonstrated the feasibility to fabricate 3D scaffolds with magnetic properties by combining FDM printing of a polymer blend and thermal DOD IJP of magnetic nanoparticles suspensions. In vitro tests showed proper cell adhesion and proliferation, while osteogenic differentiation studies, conducted using an external magnetic field, showed a significant increase in markers of bone formation and mineralisation. Therefore, these 3D magnetic scaffolds could be useful to remotely treat osteoporotic patients with physically disabling conditions.
As mentioned previously, the new 3D bioprinting platform was used in Chapter 4 to fabricate a drug-device prototype for preclinical assessments. The design was defined considering suggestions from clinicians and results from in silico models (e.g., degradation), as well as from the in vitro tests and in vivo assessments on a small animal model. In this context, the PhD work focused on the design and fabrication of the prototype to be in vivo evaluated on a large animal model, specifically the tibia of a sheep that presented two bi-cortical defects. Starting from the surgical procedure proposed by the clinicians, the geometry and dimensions were appropriately defined to provide proper contact with bone and cover the defect site. Then, the fabrication phase was performed FDM printing a polyester-based blend filament with the BOOST 3D bioprinter. The results of preclinical investigation revealed that the drug-device product was able to promote the healing of bone defects, and therefore it could be safely used in the next clinical phase to evaluate its efficacy in humans.
As previously seen, animal models are still the gold standard for studying osteoporosis and testing new drugs against this disease. However, they are expensive and not able to accurately reproduce the in vivo conditions. Therefore, alternative methods are needed to deal with the lack of a suitable osteoporotic bone model. Recently, 3D in vitro pathological bone models have been considered to overcome the economic and ethical issues of the traditional preclinical testing methods. In this context, the last objective of the PhD project aimed to develop a 3D in vitro model of osteoporotic bone by mechano-culture a cell-seeded osteoporotic scaffold. In parallel, a physiological 3D in vitro bone model was designed. Osteoporotic and physiological scaffolds differed in architecture and mineral density to replicate the altered microstructure and composition of osteoporotic bone. The results reveal an enhanced osteogenic differentiation under dynamic conditions, with osteoporotic scaffolds giving the impression of suppressing the bone formation as in vivo. As a next step in the in vitro study of osteoporosis, Appendix A presents the use of the aforementioned in vitro bone models to study the interaction between the human gut microbiota and bone, as the former could play a key role in the onset, prevention, and treatment of this disease.
To conclude, the presented PhD study aims to apply the tools of bioengineering to find alternative and novel methods in the treatment and study of osteoporosis. In this context, a drug-device combination product was designed to fill the current clinical gaps in osteoporotic fractures and ensure a better solution. The implemented in silico degradation model and the advanced 3D bioprinting platform were an effective tool in the design and fabrication phases of a preclinical prototype. Following the same process, a drug-device product can be realized for the clinical phase and then its commercialization. With the aim of reducing the use of animal models in this context, a 3D in vitro model of osteoporotic bone was designed, which could be used in the future to study new drugs and define personalized plans in the treatment of osteoporotic patients.