Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.

Jovic, Thomas; Thomson, Emman; Jones, Nick; Thornton, Cathy; Doak, Shareen; Whitaker, Iain

doi:10.3389/fbioe.2024.1421111

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Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.

Thomas Jovic, Emman Thomson, Nick Jones

, Cathy Thornton

, Shareen Doak

, Iain Whitaker

Frontiers in Bioengineering and Biotechnology, Volume: 12

Swansea University Authors: Thomas Jovic, Emman Thomson, Nick Jones , Cathy Thornton , Shareen Doak , Iain Whitaker

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DOI (Published version): 10.3389/fbioe.2024.1421111

Abstract

The ability to bioprint facial cartilages could revolutionise reconstructive surgery, but identifying the optimum cell source remains one of the great challenges of tissue engineering. Tissue specific stem cells: chondroprogenitors, have been extracted previously using preferential adhesion to fibro...

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Published in:	Frontiers in Bioengineering and Biotechnology
ISSN:	2296-4185
Published:	Frontiers Media S.A. 2024
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Tissue specific stem cells: chondroprogenitors, have been extracted previously using preferential adhesion to fibronectin based on the expression of CD49e: a perceived chondroprogenitor stem cell marker present on <1% of cartilage cells. This study sought to determine whether these fibronectin-adherent chondroprogenitor cells could be exploited for cartilage tissue engineering applications in isolation, or combined with differentiated chondrocytes. Nasoseptal cartilage samples from 20 patients (10 male, 10 female) were digested to liberate cartilage-derived cells (CDCs) from extracellular matrix. Total cell number was counted using the Trypan Blue exclusion assay and added to fibronectin coated plates for 20 min, to determine the proportion of fibronectin-adherent (FAC) and non-adherent cells (NFACs). All populations underwent flow cytometry to detect mesenchymal stem/progenitor cell markers and were cultured in osteogenic, chondrogenic and adipogenic media to determine trilineage differentiation potential. Cell adherence and growth kinetics of the different populations were compared using iCELLigence growth assays. Chondrogenic gene expression was assessed using RT-qPCR for Type 2 collagen, aggrecan and SOX9 genes. Varying proportions of NFAC and FACs were cultured in alginate beads to assess tissue engineering potential. 52.6% of cells were fibronectin adherent in males and 57.7% in females, yet on flow cytometrical analysis, only 0.19% of cells expressed CD49e. Moreover, all cells (CDC, FAC and NFACs) demonstrated an affinity for trilineage differentiation by first passage and the expression of stem/progenitor cell markers increased significantly from digest to first passage (CD29, 44, 49e, 73 and 90, p < 0.0001). No significant differences were seen in adhesion or growth rates. Collagen and aggrecan gene expression was higher in FACs than CDCs (2-fold higher, p = 0.008 and 0.012 respectively), but no differences in chondrogenic potential were seen in any cell mixtures in 3D culture models. The fibronectin adhesion assay does not appear to reliably isolate a chondroprogenitor cell population from nasoseptal cartilage, and these cells confer no advantageous properties for cartilage tissue engineering. Refinement of cell isolation methods and chondroprogenitor markers is warranted for future nasoseptal cartilage tissue engineering efforts. [Abstract copyright: Copyright © 2024 Jovic, Thomson, Jones, Thornton, Doak and Whitaker.]</abstract><type>Journal Article</type><journal>Frontiers in Bioengineering and Biotechnology</journal><volume>12</volume><journalNumber/><paginationStart/><paginationEnd/><publisher>Frontiers Media S.A.</publisher><placeOfPublication/><isbnPrint/><isbnElectronic/><issnPrint/><issnElectronic>2296-4185</issnElectronic><keywords>Chondroprogenitor, cartilage, tissue engineering, fibronectin, stem cell</keywords><publishedDay>26</publishedDay><publishedMonth>9</publishedMonth><publishedYear>2024</publishedYear><publishedDate>2024-09-26</publishedDate><doi>10.3389/fbioe.2024.1421111</doi><url/><notes/><college>COLLEGE NANME</college><department>Medical School</department><CollegeCode>COLLEGE CODE</CollegeCode><DepartmentCode>MEDS</DepartmentCode><institution>Swansea University</institution><apcterm>SU College/Department paid the OA fee</apcterm><funders>The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by The Scar Free Foundation and Health and Care Research Wales Programme of research in Reconstructive Surgery and Regenerative Medicine, which has been established in the ReconRegen Research Centre at Swansea University in partnership with Swansea Bay University Health Board. Additionally, TJ would like to acknowledge funding from Action Medical Research and the VTCT Foundation (GN2782) and Microtia United Kingdom. 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spelling	v2 68056 2024-10-24 Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes. 7d95ed2bceb18fc0fdfd4048277c6eed Thomas Jovic Thomas Jovic true false dc9e7718f6f8bb11d3df1d0cd8245318 Emman Thomson Emman Thomson true false 0fce0f7ddbdbfeb968f4e2f1e3f86744 0000-0003-4846-5117 Nick Jones Nick Jones true false c71a7a4be7361094d046d312202bce0c 0000-0002-5153-573X Cathy Thornton Cathy Thornton true false 8f70286908f67238a527a98cbf66d387 0000-0002-6753-1987 Shareen Doak Shareen Doak true false 830074c59291938a55b480dcbee4697e Iain Whitaker Iain Whitaker true false 2024-10-24 MEDS The ability to bioprint facial cartilages could revolutionise reconstructive surgery, but identifying the optimum cell source remains one of the great challenges of tissue engineering. Tissue specific stem cells: chondroprogenitors, have been extracted previously using preferential adhesion to fibronectin based on the expression of CD49e: a perceived chondroprogenitor stem cell marker present on <1% of cartilage cells. This study sought to determine whether these fibronectin-adherent chondroprogenitor cells could be exploited for cartilage tissue engineering applications in isolation, or combined with differentiated chondrocytes. Nasoseptal cartilage samples from 20 patients (10 male, 10 female) were digested to liberate cartilage-derived cells (CDCs) from extracellular matrix. Total cell number was counted using the Trypan Blue exclusion assay and added to fibronectin coated plates for 20 min, to determine the proportion of fibronectin-adherent (FAC) and non-adherent cells (NFACs). All populations underwent flow cytometry to detect mesenchymal stem/progenitor cell markers and were cultured in osteogenic, chondrogenic and adipogenic media to determine trilineage differentiation potential. Cell adherence and growth kinetics of the different populations were compared using iCELLigence growth assays. Chondrogenic gene expression was assessed using RT-qPCR for Type 2 collagen, aggrecan and SOX9 genes. Varying proportions of NFAC and FACs were cultured in alginate beads to assess tissue engineering potential. 52.6% of cells were fibronectin adherent in males and 57.7% in females, yet on flow cytometrical analysis, only 0.19% of cells expressed CD49e. Moreover, all cells (CDC, FAC and NFACs) demonstrated an affinity for trilineage differentiation by first passage and the expression of stem/progenitor cell markers increased significantly from digest to first passage (CD29, 44, 49e, 73 and 90, p < 0.0001). No significant differences were seen in adhesion or growth rates. Collagen and aggrecan gene expression was higher in FACs than CDCs (2-fold higher, p = 0.008 and 0.012 respectively), but no differences in chondrogenic potential were seen in any cell mixtures in 3D culture models. The fibronectin adhesion assay does not appear to reliably isolate a chondroprogenitor cell population from nasoseptal cartilage, and these cells confer no advantageous properties for cartilage tissue engineering. Refinement of cell isolation methods and chondroprogenitor markers is warranted for future nasoseptal cartilage tissue engineering efforts. [Abstract copyright: Copyright © 2024 Jovic, Thomson, Jones, Thornton, Doak and Whitaker.] Journal Article Frontiers in Bioengineering and Biotechnology 12 Frontiers Media S.A. 2296-4185 Chondroprogenitor, cartilage, tissue engineering, fibronectin, stem cell 26 9 2024 2024-09-26 10.3389/fbioe.2024.1421111 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University SU College/Department paid the OA fee The author(s) declare that financial support was received for the research, authorship, and/or publication of this article. This study was supported by The Scar Free Foundation and Health and Care Research Wales Programme of research in Reconstructive Surgery and Regenerative Medicine, which has been established in the ReconRegen Research Centre at Swansea University in partnership with Swansea Bay University Health Board. Additionally, TJ would like to acknowledge funding from Action Medical Research and the VTCT Foundation (GN2782) and Microtia United Kingdom. TJ and ET would like to acknowledge funding from the Welsh Clinical Academic Training Programme. 2024-10-24T13:50:36.7513403 2024-10-24T11:53:38.1015690 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Thomas Jovic 1 Emman Thomson 2 Nick Jones 0000-0003-4846-5117 3 Cathy Thornton 0000-0002-5153-573X 4 Shareen Doak 0000-0002-6753-1987 5 Iain Whitaker 6 68056__32699__8e6fd975deb04618b6f2ac5bdf810584.pdf 68056.VOR.pdf 2024-10-24T13:43:36.2955436 Output 6614266 application/pdf Version of Record true © 2024 Jovic, Thomson, Jones, Thornton, Doak and Whitaker. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). true eng http://creativecommons.org/licenses/by/4.0/
title	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.
spellingShingle	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes. Thomas Jovic Emman Thomson Nick Jones Cathy Thornton Shareen Doak Iain Whitaker
title_short	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.
title_full	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.
title_fullStr	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.
title_full_unstemmed	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.
title_sort	Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.
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author_id_fullname_str_mv	7d95ed2bceb18fc0fdfd4048277c6eed_*_Thomas Jovic dc9e7718f6f8bb11d3df1d0cd8245318__Emman Thomson 0fce0f7ddbdbfeb968f4e2f1e3f86744__Nick Jones c71a7a4be7361094d046d312202bce0c__Cathy Thornton 8f70286908f67238a527a98cbf66d387__Shareen Doak 830074c59291938a55b480dcbee4697e_*_Iain Whitaker
author	Thomas Jovic Emman Thomson Nick Jones Cathy Thornton Shareen Doak Iain Whitaker
author2	Thomas Jovic Emman Thomson Nick Jones Cathy Thornton Shareen Doak Iain Whitaker
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department_str	Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
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description	The ability to bioprint facial cartilages could revolutionise reconstructive surgery, but identifying the optimum cell source remains one of the great challenges of tissue engineering. Tissue specific stem cells: chondroprogenitors, have been extracted previously using preferential adhesion to fibronectin based on the expression of CD49e: a perceived chondroprogenitor stem cell marker present on <1% of cartilage cells. This study sought to determine whether these fibronectin-adherent chondroprogenitor cells could be exploited for cartilage tissue engineering applications in isolation, or combined with differentiated chondrocytes. Nasoseptal cartilage samples from 20 patients (10 male, 10 female) were digested to liberate cartilage-derived cells (CDCs) from extracellular matrix. Total cell number was counted using the Trypan Blue exclusion assay and added to fibronectin coated plates for 20 min, to determine the proportion of fibronectin-adherent (FAC) and non-adherent cells (NFACs). All populations underwent flow cytometry to detect mesenchymal stem/progenitor cell markers and were cultured in osteogenic, chondrogenic and adipogenic media to determine trilineage differentiation potential. Cell adherence and growth kinetics of the different populations were compared using iCELLigence growth assays. Chondrogenic gene expression was assessed using RT-qPCR for Type 2 collagen, aggrecan and SOX9 genes. Varying proportions of NFAC and FACs were cultured in alginate beads to assess tissue engineering potential. 52.6% of cells were fibronectin adherent in males and 57.7% in females, yet on flow cytometrical analysis, only 0.19% of cells expressed CD49e. Moreover, all cells (CDC, FAC and NFACs) demonstrated an affinity for trilineage differentiation by first passage and the expression of stem/progenitor cell markers increased significantly from digest to first passage (CD29, 44, 49e, 73 and 90, p < 0.0001). No significant differences were seen in adhesion or growth rates. Collagen and aggrecan gene expression was higher in FACs than CDCs (2-fold higher, p = 0.008 and 0.012 respectively), but no differences in chondrogenic potential were seen in any cell mixtures in 3D culture models. The fibronectin adhesion assay does not appear to reliably isolate a chondroprogenitor cell population from nasoseptal cartilage, and these cells confer no advantageous properties for cartilage tissue engineering. Refinement of cell isolation methods and chondroprogenitor markers is warranted for future nasoseptal cartilage tissue engineering efforts. [Abstract copyright: Copyright © 2024 Jovic, Thomson, Jones, Thornton, Doak and Whitaker.]
published_date	2024-09-26T13:50:35Z
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score	11.0351515

Nasoseptal chondroprogenitors isolated through fibronectin-adherence confer no biological advantage for cartilage tissue engineering compared to nasoseptal chondrocytes.

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