Spatiotemporal control of siponimod delivery for the regeneration of critical bone defects

Abstract

Tissue engineering aims at regenerating damaged tissue by using synthetic or natural materials and has applications across the different tissue types, including bone. A major challenge in bone tissue engineering includes the availability of materials that possess desirable properties including osteogenic potential, as well as osteoconductive and osteoinductive features to support bone regeneration, this challenge is magnified in the case of critical bone defects. The gold-standard treatment for such defects is autologous bone grafting, which suffers from issues related to the availability of material and the morbidity associated with surgeries to harvest the tissue. Therefore, it is important to consider alternative materials and therapeutic options that may contribute to improving the outcomes of bone tissue engineering issues. The overall aim of this thesis was to investigate the therapeutic potential of the small drug molecule, siponimod, to influence key cell process inherent in bone regeneration and to investigate the formulation design, functionality and regenerative potential of suitable scaffold constructs that exert spatiotemporal control over siponimod delivery both in vitro and in an in vivo critical defect model. Chapter 1 provided a general introduction of key concepts discussed throughout the thesis including a background on bone anatomy and biology. Thereafter, the chapter introduced tissue engineering in general with a focus on intrinsic aspects of bone tissue engineering namely scaffolds, cells, and signals. It described the key requirements in the design of scaffolds suitable for bone regeneration and provided an overview of the materials and techniques used in the fabrication of scaffolds for bone tissue engineering. In particular it highlighted, the materials used throughout this thesis, such as the natural and synthetic polymers collagen and poly lactide-co-glycolide (PLGA) used in Chapter 4 & 5, and the bioactive ceramic hydroxyapatite (HA) used in Chapter 5. This first chapter also addressed the use of protein and small molecule signal therapeutics in bone tissue engineering, including a brief introduction of sphingosine 1-phosphate. Chapter 2 thus followed with an in-depth review of the role of sphingosine 1-phosphate (S1P) in bone biology and its potential therapeutic use in bone repair. The role of S1P in nervous, cardiovascular, and immune systems is well established, however, knowledge regarding its role in bone biology and the utility of specific S1P receptor modulation in bone repair was lacking. Therefore, Chapter 2 not only aimed to add to the available literature on the role of S1P signalling in bone repair, but also to contribute to the identification of S1P mediated processes that could be targeted therapeutically. The culmination of the review in Chapter 2 was the selection of S1P1 receptor modulation as a target, and siponimod as a selective agonist for further investigation. Thereafter, Chapter 3 investigated the in vitro bone regenerative potential of the S1P receptor modulator, siponimod. Specifically, it aimed to identify the impact of siponimod on key cellular processes including cell viability, proliferation, differentiation and migration using human foetal osteoblasts (hFOB), as well as cell proliferation and migration using human umbilical vein endothelial cells (HUVEC). The hypothesis underpinning Chapter 3 was that selective S1P1 signalling using the S1P1/5 agonist, siponimod would stimulate osteoblast proliferation, differentiation, and migration as well as endothelial cell proliferation and migration. The results of this chapter showed for the first time that siponimod indeed promotes osteoblast differentiation while having no influence on viability and proliferation. Siponimod was also shown to promote the chemokinesis of endothelial cells, whereby it interfered with cell attachment and migration in the short-term (4 hrs) and caused a delayed (8 hrs) stimulation of endothelial migration. Taken together these results suggested that siponimod was worthy of further investigation in the context of bone regeneration. However, the balance of evidence in the bone repair literature supports the use of a localised delivery approach for sphingolipids, rather than systemic administration. This was the motivation supporting the research in Chapter 4 and Chapter 5, which investigated suitable scaffold constructs for the localised delivery of siponimod. Therefore, the hypothesis underpinning Chapter 4 was that the design of a biocompatible and biodegradable polymeric scaffold would control the presentation of siponimod in a stable and functional manner at appropriate concentrations and over relevant timeframes to exploit its potential for enhanced bone regeneration. This chapter thus detailed the preparation, characterisation and in vitro assessment of PLGA-based electrospun material coupled with collagen and loaded with siponimod at different concentrations (0.5-2 % w/w). The physicochemical characteristics including drug stability in the solid and liquid state as well as drug loading and release properties were investigated. Additionally, in vitro cell-based investigations were carried out on the electrospun material to assess its compatibility with the cellular populations of interest, hFOB and HUVEC, and whether the released siponimod maintained the functional effects determined in Chapter 3. Results confirmed our hypothesis that siponimod could be successfully loaded with high efficiencies (80-94 %) and its release could be controlled in a stable manner (> 3 months), which was in line with a planned 12-week in vivo cranial defect study. Furthermore, the released siponimod maintained its differentiation and migration effects on hFOB and HUVEC in vitro. The scaffolds were then implanted in rat critical cranial defects for 12-weeks to assess in vivo effectiveness of the siponimod loaded scaffold. Results showed that while there was some reduction in defect size, there was no statistically significant differences between the experimental groups regarding the histomorphometrically determined area of mineralisation within the defect space. The scaffold described in Chapter 5 was designed contemporaneously with the electrospun scaffold in Chapter 4, although only the latter design was progressed to the stage of in vivo analysis. Acknowledging this, Chapter 5 provided a preliminary description of the design and characterisation of another scaffold design using electrospray-microparticles loaded with siponimod. As with the electrospun scaffold, this alternative design is based on a similar hypothesis that localised delivery of siponimod for bone regeneration was superior to systemic delivery. The microparticles were mould compressed with HA, the calcium phosphate mineral reminiscent of that found in native bone tissue, and a porogen, prior to high-pressure carbon dioxide (CO2) foaming and porogen leaching. The morphological properties of the microparticles and the completed scaffolds were assessed using scanning electron microscopy (SEM). Physicochemical properties investigated included porosity, mechanical properties, siponimod drug loading and release, and cell culture studies to assess the scaffold’s effect on hFOB and HUVEC. Results of SEM showed that the diameters of microparticles was increased by the inclusion of siponimod, while scaffolds possessed a highly porous internal structure, with morphology affected by the inclusion of HA. Drug loading efficiency was lower than those seen in Chapter 4, which was expected due to the method employed, although drug release was still sustained over 3 months. The scaffolds were found to be compatible with hFOB and HUVEC, whether seeded in direct or indirect contact with scaffolds and no significant changes to cell metabolic activity were observed. In conclusion, this thesis showed that S1P receptors have a clear impact on the biology of bone repair, with novel findings contributing to our understanding of siponimod’s in vitro effect on osteoblasts and endothelial cells, which could lead to siponimod-based therapeutic options for bone and other tissue regeneration applications. This thesis also detailed the first designs of controlled release scaffolds for siponimod, with the S1P1 agonist successfully incorporated into two different scaffolds using both electrospinning and electrospraying production methods, which enabled constructs with different compositions and physical properties to be designed. Although in vivo results of cranial defect studies did not provide statistically significant evidence of improved bone regeneration, both scaffold designs demonstrated promising cell compatibility and drug release properties, that can be further optimised to fully utilise the bone regenerative potential of siponimod and other S1P agonists.