Fabrication and Optimisation of Collagen Scaffolds for Muscle Cell-Supported Angiogenesis

Abstract

Vascularisation is essential for the success of tissue engineered scaffolds, facilitating appropriate oxygen, nutrient, and waste transport in structures larger than diffusion transport can achieve. Combining insights and priorities from the fields of tissue engineering and cultivated meat, this Thesis describes the development of 3D collagen scaffolds capable of supporting differentiated muscle cell populations with the potential for vascularisation. Collagen I scaffolds with varying internal architectures were fabricated and characterised, cell-material interactions were assessed in both 2D and 3D, and human dermal microvascular endothelial cell (HDMEC) behaviour was evaluated in human skeletal muscle cell (HSkMC)-seeded scaffolds. The internal architecture of porous scaffolds is known to influence cell outcomes; therefore, control of structure via the fabrication process is highly desirable. The first stage of this work investigated the impact of collagen slurry composition on the final scaffold architecture for freeze-dried structures. A systematic approach was employed to deconvolute the influence of protonation and acid type on the final collagen scaffold structure, including pore size, shape, and distribution. The relationship between pH and architectural properties was found to deviate from linearity due to the presence and influence of conjugate base and undissociated acid species. This work provided an additional mechanism for architectural control of freeze-dried scaffolds without the need for extensive thermal calibration and allowed collagen scaffolds containing specific architectures to be fabricated for subsequent investigation. Collagen fibres offer cell binding motifs along their surface; however, the behaviour of human muscle cells on collagen substrates requires further exploration. Hence, physio-chemical optimisation of 2D collagen I films (as a proxy for pore walls) was conducted for HSkMCs. It was found that selective attachment occurred via β1-containing integrins. The relevant amino acid motifs were ablated by EDC/NHS crosslinking, although the mechanical stability imbued by crosslinking improved proliferation for up to 30% of the standard crosslinking degree reported in literature. However, while 2D collagen films supported early muscle cell culture, they were unsuitable for cell adhesion longer-term. Coating with an Ile-Lys-Val-Ala-Val pentapeptide (IKVAV) improved cell stability and elongation; eccentricity was identified as an early predictor of proliferative success for HSkMCs. Following surface optimisation for HSkMCs, the internal architecture of 3D collagen scaffolds was probed for its influence on HSkMC behaviour. The importance of pore distribution in scaffold infiltration was identified, with scaffolds fabricated from collagen hydrated in acetic acid at pH 3.0 resulting in an average pore size c.120 μm and fully-occupied pores after 10 days of culture. Additionally, isotropic pores enabled more rapid infiltration of large scaffolds, whereas anisotropic pores were found to promote cell elongation and alignment at the expense of migration. Due to these advantages in terms of cell migration, isotropic scaffolds were selected for further investigation in this project. HSkMCs were also shown to secrete vascular endothelial growth factor (VEGF) in 3D in vitro cultures, which is a key growth factor in the stimulation of angiogenic behaviour. To validate the hypothesis that the HSkMC-supplied VEGF would improve cell outcomes in co-cultures compared to monocultures, empty and HSkMC-loaded collagen scaffolds were investigated for their capacity to support HDMECs. The longterm presence of HDMECs was enhanced for up to 21 days in co-culture with differentiated HSkMCs, and platelet endothelial cell adhesion molecule-1 (PECAM-1) assembly, which is indicative of the beginnings of vascular network formation, was upregulated. The ratio of HSkMCs to HDMECs was found to be critical for the promotion of stable, interconnected endothelial structures, with additional support cells resulting in increased PECAM-1 expression by endothelial cells. The implications of this research span both theoretical and applied domains. By separating the effects of acid type and pH on collagen scaffold structures, this work broadens the design space for freeze-dried scaffolds for tissue engineering. Additionally, mechanisms underpinning HSkMC-collagen interactions have been investigated, and the relationship between HSkMC morphology and proliferation introduced a potential metric for rapid surface evaluation. HSkMCs hosted in the developed scaffolds were demonstrated to secrete VEGF; the ability of these 3D collagen scaffolds to support both muscle and endothelial cells underscores their potential for vascularised engineered tissues, contributing to advancements in both regenerative medicine and, potentially, the scalability of cultivated meat production.