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Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering / HON COURCEY

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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...

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Published: Swansea University 2026
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
first_indexed 2026-02-17T12:15:19Z
last_indexed 2026-02-18T05:35:32Z
id cronfa71444
recordtype RisThesis
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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)&gt;TEMPO-mediated oxidised(TTC)&gt;ETC on D1-7 and TTC &gt;CTC&gt;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. 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S.</supervisor><degreelevel>Doctoral</degreelevel><degreename>Ph.D</degreename><degreesponsorsfunders>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</degreesponsorsfunders><apcterm/><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</funders><projectreference/><lastEdited>2026-02-17T12:20:17.3932208</lastEdited><Created>2026-02-17T12:07:11.8297046</Created><path><level id="1">Faculty of Medicine, Health and Life Sciences</level><level id="2">Swansea University Medical School - Biomedical Science</level></path><authors><author><firstname>HON</firstname><surname>COURCEY</surname><order>1</order></author></authors><documents><document><filename>71444__36248__bfd0ae4bbfc84c00a2a47560cf289932.pdf</filename><originalFilename>2026_De_Courcey_C.final.71444.pdf</originalFilename><uploaded>2026-02-17T12:13:10.4278739</uploaded><type>Output</type><contentLength>57515528</contentLength><contentType>application/pdf</contentType><version>E-Thesis &#x2013; open access</version><cronfaStatus>true</cronfaStatus><documentNotes>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).</documentNotes><copyrightCorrect>true</copyrightCorrect><language>eng</language><licence>https://creativecommons.org/licenses/by-nc-nd/4.0/</licence></document></documents><OutputDurs/></rfc1807>
spelling 2026-02-17T12:20:17.3932208 v2 71444 2026-02-17 Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering d5b2ca881ad408f28c63c64a339519a2 HON COURCEY HON COURCEY true false 2026-02-17 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. E-Thesis Swansea University Nanocellulose, cartilage tissue engineering, advanced in vitro model 20 1 2026 2026-01-20 10.23889/SUThesis.71444 COLLEGE NANME COLLEGE CODE Swansea University Clift, M. J. D., and Whitaker, I. S. Doctoral Ph.D 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 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 2026-02-17T12:20:17.3932208 2026-02-17T12:07:11.8297046 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science HON COURCEY 1 71444__36248__bfd0ae4bbfc84c00a2a47560cf289932.pdf 2026_De_Courcey_C.final.71444.pdf 2026-02-17T12:13:10.4278739 Output 57515528 application/pdf E-Thesis – open access true 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). true eng https://creativecommons.org/licenses/by-nc-nd/4.0/
title Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
spellingShingle Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
HON COURCEY
title_short Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
title_full Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
title_fullStr Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
title_full_unstemmed Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
title_sort Establishing the effects of Nanocellulose-based bio-inks in an advanced 3D in vitro model for cartilage tissue engineering
author_id_str_mv d5b2ca881ad408f28c63c64a339519a2
author_id_fullname_str_mv d5b2ca881ad408f28c63c64a339519a2_***_HON COURCEY
author HON COURCEY
author2 HON COURCEY
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publishDate 2026
institution Swansea University
doi_str_mv 10.23889/SUThesis.71444
college_str Faculty of Medicine, Health and Life Sciences
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hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str 1
active_str 0
description 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.
published_date 2026-01-20T05:34:48Z
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score 11.096892

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