| dc.description.abstract | Every 3 seconds a person suffers from an osteoporosis (OP) related bone fracture, resulting in significant disability, loss of independence, and early mortality. This systemic skeletal disease is characterised by low bone mineral density inducing a predisposition to bone fragility and increased risk of fracture. OP is particularly prevalent across the elderly, with 1 in 2 women and 1 in 5 men doomed to experience a fragility fracture in their remaining lifetime after the age of 50 years. As demographics shift towards an ageing population, OP represents a tremendous societal and economic burden, with treatment costs already reaching €56.9 billion per year in the EU alone. Current treatments for bone fractures include gold standard autologous/allogenous bone grafts and plate fixation. However, these target the later repair phase of fracture healing and thus often fail to regulate the earlier inflammation phase, which is commonly disrupted in osteoporotic fractures and characterised by heightened local or systemic inflammatory levels. Similar complications can be observed in patients suffering from chronic inflammatory conditions such as diabetes, obesity, or age-related immunodeficiency. This highlights the urgent need for the development of novel treatment strategies that aim at modulating the initial inflammatory phase and its associated immune cells to create a regenerative environment bolstering fracture healing, thus addressing the shortcomings of traditional therapies.
Mechanical signals are among the most potent regulators of bone repair and may offer the potential to influence local inflammation, by acting upon the early fracture haematoma as early as in the first week post-fracture. By manipulating the plate fixator stiffness stabilising fractures, it has been shown that bone regeneration is exquisitely sensitive to loading magnitude (interfragmentary strain), where low/moderate strains accelerate healing, while high strains result in delayed/non-healing. Despite the emergence of the mechano-immunology field which aims at interrogating how local mechanical cues shape the immune cell phenotype and function, it is still unclear how mechanosignalling could be harnessed to positively mediate the early immune response and subsequent orchestration of bone healing processes. Therefore, the overarching goal of this thesis was to provide substantial insights into how different levels of mechanical loading as experienced during ambulation of a fixated large bone defect could regulate macrophage behaviour, a master regulator of the early immune response, and to identify novel small molecule therapeutics capable of targeting macrophage (mechano)signalling to modulate the damaging effects of heightened inflammation seen in various pathologies and/or excessive mechanical stimulation seen in poorly fixated fractures, to ultimately elicit a regenerative immune environment conducive to vascularisation and long-term bone regeneration.
In Chapter 3 of this thesis, a haematoma-mimetic 3D fibrin hydrogel was developed as a means to mirror the local macrophage microenvironment during the early stages of fracture healing, which current traditional in vitro culturing platforms fail to recapitulate. The integration of both local fibrin ECM composition and 3D architecture yielded an enhanced physiological macrophage behaviour capable of achieving more pronounced regenerative phenotypes and coordination of angiogenesis/osteogenesis that better mirror the healing processes as seen in vivo.
Having established a physiologically relevant 3D in vitro model of the fracture haematoma, the following Chapter 4 first sought to develop a purpose-built bioreactor that can replicate the different loading conditions (uniaxial compressive strain) experienced during ambulation of a fixated or unfixed large bone defect on the previously validated haematoma mimetic hydrogels. Harnessing this system, we then demonstrated that macrophages are mechanoresponsive and sensitive to the loading magnitude of compressive forces, responding accordingly with changes in their phenotype and secretome. Lower loading (5% strain) was capable of driving a hybrid phenotype and higher regenerative secretome in macrophages, while inhibiting inflammatory levels in pro-inflammatory M1-like macrophages, ultimately reshaping local vasculature. Conversely, higher loading (35% strain) elicited a poor regenerative secretome in macrophages which appeared detrimental not only to early vascularisation but also to later stage mineralisation. The results of this study thus highlight controlled mechanics as a potential novel approach to modulate heightened inflammation commonly expressed in compromised bone defects.
In the context of complex fractures where achieving constrained mechanics remains particularly challenging, we explored in Chapter 5 an alternative treatment strategy by therapeutically targeting cAMP macrophage signalling utilising the cAMP upstream activator Forskolin. Treatment of unactivated macrophages with Forskolin elicited a hybrid macrophage phenotype generating a highly pro-regenerative immune secretome considerably enhancing vascular growth and mineralisation. Additionally, in the context of severe inflammation as experienced in compromised bone defects, Forskolin treatment was capable of significantly inhibiting inflammatory levels in a pro-inflammatory M1-like macrophage, while inducing robust increases in pro-regenerative cytokine release, which slightly enhanced vascular growth. Together, these findings show the tremendous potential of targeting macrophage cAMP signalling pharmacologically as a complimentary approach to controlled mechanics for the treatment of challenging fractures.
In Chapter 6, we inquired whether pharmacological targeting of the mechanosensitive calcium-dependent TRPV4 channel, using the newly identified TRPV4 antagonist GSK2798745, could help combat heightened inflammatory levels commonly experienced in mechanically unstable fractures. We reported a reduction in macrophage inflammatory levels upon GSK2798745 treatment, which ultimately moderately enhanced vascularisation without affecting long-term bone deposition. We next developed a novel method for GSK2798745 encapsulation into PLGA nanoparticles with the aim of creating a sustainable drug delivery platform.
In Chapter 7, we further highlighted the potential of targeting macrophage cAMP signalling pharmacologically with the cAMP upstream activator Forskolin, which we have shown in Chapter 5 to dampen inflammatory levels in a traditional 2D in vitro culturing platform, in mitigating the detrimental effects of heightened inflammation and excessive compressive loading commonly experienced in challenging factures, utilising our previously developed fracture haematoma-like in vitro platform, on the modulation of the initial immune response and subsequent healing processes. We first demonstrated that Forskolin was capable of attenuating inflammation in an overly expressed inflammatory immune response in a cytokine-dependent manner, while promoting a regenerative immune secretome, which was not seen to be detrimental to vascular growth, promoting a healing progression towards endochondral ossification. Furthermore, Forskolin was capable of enhancing the regenerative secretome of macrophages subjected to higher loading (35% strain), ultimately promoting the establishment of the early vasculature and initiating intramembranous ossification.
In conclusion, this study provides compelling evidence that local mechanical cues possess potent immunomodulatory effects on the early immune response, which not only contributes to our understanding of how local interfragmentary strain influences short- and long-term fracture healing, but also demonstrates a novel approach to regulate the early immune response in bone defects, by achieving local controlled mechanics post-fracture. Furthermore, we provided alternative treatment strategies for challenging fractures by targeting pharmacologically macrophage (mechano)signalling as a means to reverse the detrimental effects of heightened inflammation or abnormal loading conditions commonly seen in compromised or unstable fractures. Taken together, this data will inform the development of novel mechano-immunomodulatory strategies to enhance bone regeneration. | en |
