Spontaneous 3D Micropatterning of BMP-2 in Self-assembling Nanoclay Gel

Abstract

Limitations of current clinical treatments for critical size bone defects have motivated the development of new strategies for bone tissue engineering. In this regard, emulating the three dimensional (3D) hierarchical organisation of biochemical cues found in the native cellular microenvironment is likely to be key to generate anisotropic biomaterials with distinct levels of functionality. However, despite advances in tissue engineering (TE), achieving stable structures incorporating 3D micropatterning of biochemical cues, particularly that preserve resolution with an increase in size has proven challenging. Self-assembling nanoclay-gels have established potential in TE due to their capacity to sequester proteins for sustained localised bioactivity. This thesis explores the hypothesis that bottom-up selfassembly of nanoclay/protein structures can be harnessed to achieve a gradient of proteins, allowing the delivery of localised spatio-temporal niches for enhanced bone tissue regeneration. Addition of nanoclay-gels into high concentration protein solutions promoted self-assembly of a structure with 3D micropatterning of proteins. Polarised light and scanning electron microscopy confirmed that a reaction-diffusion process was responsible for the scaffold assembly, where the proteins (or gelator) reacted with the clay nanoparticles and simultaneously diffused through the clay-gel in a concentration-dependent manner. The process led to the formation of a proteinclay complex structure with a periodical arrangement. Furthermore, confocal images demonstrated that the diffusion front or the interface between the reacted and unreacted clay-gel region was responsible for the 3D localisation of loaded proteins. By changing the assembly parameters, such as concentration, ionic strength, incubation time and temperature. Also, the solute size and net charge it was possible to control the diffusion coefficient of the assembly solution, and as a result, the localisation of proteins loaded. Tuning of the assembly and loading process allowed the generation of scaffolds with punctuated or gradual gradients of different proteins. Also, the versatility of the system supported the assembly of structures at scale with a range of dimensions (0.2 to 1 mm) and shapes (droplets, cylinders and strings) while preserving the resolution of protein patterning. Finally, a subcutaneous mouse model revealed that the punctuated localisation of BMP-2 inside the scaffold had the potential to control the spatio-temporal formation of mature bone. Thus, this novel system provides the opportunity to design customised scaffolds with complex biochemical gradients, dimensions and shapes for bone with clinical relevance.

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Identifiers

ORCID for Jonathan Dawson: orcid.org/0000-0002-6712-0598

ORCID for Nicholas Evans: orcid.org/0000-0002-3255-4388

ORCID for Richard Oreffo: orcid.org/0000-0001-5995-6726

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