E-Thesis 529 views 10 downloads
Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering / HON COURCEY
Swansea University Author: HON COURCEY
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PDF | E-Thesis – open access
Copyright: the author, Cynthia de Courcey, 2026. Distributed under the terms of a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License (CC BY-NC-ND 4.0).
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DOI (Published version): 10.23889/SUThesis.71444
Abstract
Trauma, cancer or congenital conditions can lead to the loss of facial cartilage resulting in altered function and form. Tissue engineering (3D-printing) has the potential to create bespoke cartilage implants for reconstruction with biomaterials as ink carriers loaded with patient cells. There is ri...
| Published: |
Swansea University
2026
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| Institution: | Swansea University |
| Degree level: | Doctoral |
| Degree name: | Ph.D |
| Supervisor: | Clift, M. J. D., and Whitaker, I. S. |
| URI: | https://cronfa.swan.ac.uk/Record/cronfa71444 |
| Abstract: |
Trauma, cancer or congenital conditions can lead to the loss of facial cartilage resulting in altered function and form. Tissue engineering (3D-printing) has the potential to create bespoke cartilage implants for reconstruction with biomaterials as ink carriers loaded with patient cells. There is rising interest in nanocellulose as a bio-ink candidate, but relative paucity of studies investigating an all-natural hybrid bio-ink inclusive of alginate (most common natural bio-ink material) and hyaluronic acid (HA)(native component of human extra-cellular matrix). This study aims to: i) evaluate the cytotoxic and pro-inflammatory effects of each bio-ink components individually and combined as bio-inks; ii) create an advanced 3D in vitro model for biocompatibility testing; and iii) apply the model for toxicity testing, refinement of bio-ink formulations and further model characterisation with differential gene expression analysis.Six forms of nanocellulose, alginate, HA, and two cross-linker agents were investigated for their biological impact to human chondrocyte C20A4 and dermal fibroblast HFF-1 cell lines. Material sterility was determined by microbial growth assay. Cell-line characterisation and material exposures (21 days) and cross-linker exposures (7 days) were examined for cell viability, morphology and pro-inflammatory mediators (IL-6/IL-8) release. Minimum cross-linkage time and rheologically defined gelation behaviour of bio-inks with calcium chloride (CaCl2)permitted completion of a standard operating procedure for the creation of an advanced 3D in vitro model. To mimic the implanted in vivo scenario, the in vitro model consisted of chondrocyte-encapsulated and fibroblast surface-seeded cross-linked hydrogels. RNA extraction via the spin column technique with optimisations were performed. Differential gene expression analysis was conducted using Nanostring against the nanocellulose-based bio-ink with the 3D in vitro model.All materials maintained sterility over 21 days, except for pulp-derived nanocellulose which were excluded. Material exposures (chondrocytes: nanocellulose/alginate/HA;fibroblasts: alginate/HA) showed no significant cytotoxic effects over 21 days.Enzymatically pretreated nanocellulose (ETC) displayed the lowest pro-inflammatory effects when exposed against chondrocytes, with trends of carboxymethylated(CTC)>TEMPO-mediated oxidised(TTC)>ETC on D1-7 and TTC >CTC>ETC on D14-21.CaCl2 exposure on fibroblasts demonstrated a dose- and time-dependent cytotoxicity supporting use of the lowest concentration and shortest cross-linkage time to minimise adverse cellular impact. Testing of optimised bio-inks (ETC:Alginate and ETC: Alginate:HA at 6mg/ml in media) demonstrated that nanocellulose-based bio-ink inclusive of HA was superior for chondrocyte proliferation. Heightened IL-6/IL-8 and lactate dehydrogenase release at latter timepoints indicated the need for further model development. Nanostring was successfully applied for differential gene expression analysis, which corroborated pro-inflammatory effects observed via cytokine quantification, as well as identified multiple areas of interest for further research.Overall, ETC was shown to be a promising bio-ink candidate, and when combined with alginate and HA, formed a complete bio-ink formulation specific for 3D-bioprinted cartilage constructs for reconstructive purposes. Biological and rheological testing identified optimal parameters for bio-ink and in vitro model creation, whilst RNA extraction from chondrocytes encapsulated within cross-linked bio-ink was feasible and applicable with the Nanostring technology. |
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| Keywords: |
Nanocellulose, cartilage tissue engineering, advanced in vitro model |
| College: |
Faculty of Medicine, Health and Life Sciences |
| Funders: |
Joint Royal College of Surgeons (RCS), Blond McIndoe Research Foundation One Year Surgical Research Fellowship, Joint Action Medical Research (AMR), and VTCT Foundation Research Training
Fellowship |