Development of an integrated, printed cell-scaffold construct for bone repair

Abstract

The rising incidence of bone disorders, exacerbated by an increasing ageing population worldwide, has resulted in an unmet need for more effective therapies. Bone tissue engineering is seen as a means of developing alternatives to conventional bone grafts for the repair or reconstruction of bone defects through the utilisation of biomaterials, cells, and signalling factors. However, skeletal tissue engineering faces several challenges in order to achieve full translation into clinical practice. The use of additive manufacturing techniques to biofabricate bone offers one potential solution, with its inherent capability for reproducibility, accuracy and customisation of scaffolds, as well as the potential capability for cell and signalling factor delivery. This thesis outlines the approach taken to develop such an integrated construct, which could possibly be used for bone repair. Chapter 1 begins by providing an overview of the current understanding of bone biology and the factors involved in bone repair, before proceeding to examine the current state of bone biofabrication and the necessary factors for success, while discussing the key issues limiting its use to date. The chapter concludes by stressing the need for standardisation of methods for bioprinting, in vitro and in vivo approaches and analyses, and calls for improvements to design and bioprinting software if biofabrication is to achieve its potential for clinical translation. Chapter 2 summarises the core materials and methods used throughout the project. The next three chapters describe the process leading up to the creation of a biofabricated construct through harnessing the osteogenic capacity of STRO-1 enriched bone marrow stromal cells and biocompatible materials. Chapter 3 describes established isolation and culture protocols for STRO-1 enriched bone marrow stromal cells and confirms that STRO-1 enriched bone marrow stromal cells are induced to undergo osteogenic differentiation by 1,25-dihydroxyvitamin D3 stimulation, and when seeded onto microporous, micro-rough titanium templates, despite an inverted culture approach. The results suggest STRO-1 enriched bone marrow stromal cells are a suitable cell type for use in bone repair. Chapter four details the various combinations, and potential, of multi-material bioinks for cell encapsulation and delivery purposes. Bioinks, composed of chemically cross-linked, 4% w/v low viscosity alginate, and to a lesser extent, gelatin containing 1% w/v hyaluronic acid, were found to be suitable for STRO-1 enriched bone marrow stromal cell delivery, demonstrating cell viability and proliferation post-seeding. Chapter 4 also describes the methods used in the design, generation, and characterisation of novel polycaprolactone-based scaffolds manufactured by a three-dimensional printer. Although polycaprolactone has been utilised in biomedical devices for decades, the inherent hydrophobicity of polycaprolactone has limited its use as a scaffold for cell seeding, usually requiring post-processing steps to resolve this problem. In this project, non-treated, porous, cylindrical polycaprolactone-based scaffolds were not only reproducibly manufactured by melt extrusion printing with high resolution, but were also shown to be biocompatible, with an average total porosity of 52%. Microcomputerised tomography image reconstruction analyses further revealed that 99.5% of the created pores were interconnected. Chapter 5 reports the creation of an integrated construct potentially suitable for bone reparation purposes. A manual aerosol spray was used to coat printed scaffolds with a biomimetic bioink. While the process did not result in an appreciable reduction in porosity, confocal light and scanning electron microscopy showed major alterations to the surface topography of the scaffolds. STRO-1 enriched bone marrow stromal cells deposited by this aerosol method onto non-treated, as well as biomimetic bioink-coated, 3D printed scaffolds demonstrated good viability, increased ALP activity, and underwent osteogenic differentiation over 21 days in vitro. Chapter 6 surmises the findings of this project, and concludes with a personal perspective on the future (potential) direction of biofabrication in bone tissue engineering.

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Identifiers

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

ORCID for Rahul Tare: orcid.org/0000-0001-8274-8837

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