Al Belushi, Hind; (2020) Development and characterisation of MSC-seeded decellularised airway scaffolds for regenerative bioengineering. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Tracheal tissue engineering (TE) is a potential solution for long tracheal lesions and recent clinical experience yielded promising results but challenges remain with respect to measurable criteria for acceptance of decellularised scaffolds, optimisation of cell seeding and understanding the biology of the seeded cells post attachment. Confirming previous data from our group, I showed cellular clearance of DC scaffolds and significant reduction in total DNA levels but observed retention of residual nuclear materials within hyaline cartilage and submucosa. Evaluation of extracellular matrix components demonstrated retention of collagen and glycosaminoglycan and disrupted basement membrane components. The novel use of dynamic mechanical analysis (DMA) to measure the viscoelastic properties of tracheal cartilage in addition to tensile testing, provided the first demonstration of preservation of native viscoelastic mechanical properties after decellularisation. To overcome the limitations of passive cell seeding, I conceived partial surface dehydration (PSD) conditioning of scaffolds which significantly improved cell seeding/attachment efficiency to (96.46% 1.710) and I confirmed survival of MSCs on the scaffold in vitro. Multiphoton imaging showed limited scaffold infiltration but revealed two, distinct cell morphologies dependent on the presence or absence of adventitia. These showed different RNA transcriptomic profiles and differential gene expression. Seeded MSCs upregulated transcripts of bioactive paracrine factors associated with tissue repair, including ECM remodelling, pro-angiogenesis, antifibrosis, chemoattraction and immunomodulatory properties. Cells seeded into the adventitial layer upregulated more bioactive factors and showed lower cellular stress, suggesting a favourable effect of maintaining adventitial layer. The data presented herein form a coherent series of experiments providing novel data to the field of tracheal tissue engineering which address important GMP issues such as in-process acceptance criteria for scaffolds and data to support the rationale of autologous MSC seeding prior to implantation. These results allowed us to manufacture an improved clinical product for a compassionate case.

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