Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold

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Abstract

Purpose

To utilize melt electrowriting (MEW) technology using poly-(ε-caprolactone) (PCL) coupled with a 2-step co-culturing strategy for the development of a conjunctival bi-layer synthetic construct.

Methods

Melt electrowritten scaffolds using PCL were fabricated using an in-house-built MEW printer. Human conjunctival stromal cells (CjSCs) and epithelial cells (CjECs) were isolated from donor tissue. A 2-step co-culture method was done by first seeding the CjSCs and culturing for 4 weeks to establish a stromal layer, followed by CjECs and co-culturing for 2 more weeks. Cultured cells were each characterized by morphology and marker expression on immunofluorescence and qPCR. The produced construct was assessed for cellular proliferation using viability assays. The bi-layer morphology was assessed using scanning electron microscopy (SEM), confocal microscopy, and immunofluorescence imaging. The expression of extracellular matrix components and TGF-b was evaluated using qPCR.

Results

CjSCs were spindle-shaped and vimentin + while CjECs were polygonal and CK13 + . CjSCs showed consistent proliferation and optimal adherence with the scaffold at the 4-week culture mark. A 2-layered construct consisting of a CjSC-composed stromal layer and a CjEC-composed epithelial layer was appreciated on confocal microscopy, SEM, and immunofluorescence. CjSCs secreted collagens (types I, V, VI) but at differing amounts from natural tissue while TGF-b production was comparable.

Conclusion

The 3D-printed melt electrowritten PCL scaffold paired with the 2-step co-culturing conditions of the scaffold allowed for the first approximation of a bi-layered stromal and epithelial reconstruction of the conjunctiva that can potentially improve the therapeutic arsenal in ocular surface reconstruction.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10792-022-02418-y.

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Most cited references 43

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The return of a forgotten polymer—Polycaprolactone in the 21st century

Maria Ann Woodruff, Dietmar Werner Hutmacher (2010)

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Direct writing by way of melt electrospinning.

P Dalton, Geoffrey D. Brown, Dietmar W Hutmacher (2011)

Melt electrospun fibers of poly(ϵ-caprolactone) are accurately deposited using an automated stage as the collector. Matching the translation speed of the collector to the speed of the melt electrospinning jet establishes control over the location of fiber deposition. In this sense, melt electrospinning writing can be seen to bridge the gap between solution electrospinning and direct writing additive manufacturing processes. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.

Upma Sharma, Quynh Pham, Antonios Mikos (2006)

The physical and spatial architectural geometries of electrospun scaffolds are important to their application in tissue engineering strategies. In this work, poly(epsilon-caprolactone) microfiber scaffolds with average fiber diameters ranging from 2 to 10 microm were individually electrospun to determine the parameters required for reproducibly fabricating scaffolds. As fiber diameter increased, the average pore size of the scaffolds, as measured by mercury porosimetry, increased (values ranging from 20 to 45 microm), while a constant porosity was observed. To capitalize on both the larger pore sizes of the microfiber layers and the nanoscale dimensions of the nanofiber layers, layered scaffolds were fabricated by sequential electrospinning. These scaffolds consisted of alternating layers of poly(epsilon-caprolactone) microfibers and poly(epsilon-caprolactone) nanofibers. By electrospinning the nanofiber layers for different lengths of time, the thickness of the nanofiber layers could be modulated. Bilayered constructs consisting of microfiber scaffolds with varying thicknesses of nanofibers on top were generated and evaluated for their potential to affect rat marrow stromal cell attachment, spreading, and infiltration. Cell attachment after 24 h did not increase with increasing number of nanofibers, but the presence of nanofibers enhanced cell spreading as evidenced by stronger F-actin staining. Additionally, increasing the thickness of the nanofiber layer reduced the infiltration of cells into the scaffolds under both static and flow perfusion culture for the specific conditions tested. The scaffold design presented in this study allows for cellular infiltration into the scaffolds while at the same time providing nanofibers as a physical mimicry of extracellular matrix.

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Author and article information

Contributors

Victor L. Perez: victor.perez.quinones@duke.edu

Journal

Journal ID (nlm-ta): Int Ophthalmol

Journal ID (iso-abbrev): Int Ophthalmol

Title: International Ophthalmology

Publisher: Springer Netherlands (Dordrecht )

ISSN (Print): 0165-5701

ISSN (Electronic): 1573-2630

Publication date (Electronic): 6 August 2022

Publication date PMC-release: 6 August 2022

Publication date (Print): 2023

Volume: 43

Issue: 1

Pages: 215-232

Affiliations

[1 ]GRID grid.419851.0, Department of Ophthalmology, , Ocular Surface Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, ; Miami, FL USA

[2 ]GRID grid.26790.3a, ISNI 0000 0004 1936 8606, Interdisciplinary Stem Cell Institute, , University of Miami Miller School of Medicine, ; Miami, FL USA

[3 ]GRID grid.412465.0, Department of Ophthalmology, , Second Affiliated Hospital, Zhejiang University School of Medicine, ; Hangzhou, China

[4 ]GRID grid.38142.3c, ISNI 000000041936754X, Massachusetts Eye and Ear Infirmary, , Harvard Medical School, ; Boston, MA USA

[5 ]GRID grid.26009.3d, ISNI 0000 0004 1936 7961, Department of Ophthalmology, , Foster Center for Ocular Immunology, Distinguished Stephen and Frances Foster Chair in Ocular Immunology, Duke Eye Center, ; 2351 Erwin Road, Durham, NC 27705 USA

[6 ]GRID grid.1024.7, ISNI 0000000089150953, Institute for Health and Biomedical Innovation (IHBI), , Queensland University of Technology (QUT), ; Brisbane, QLD Australia

Article

Publisher ID: 2418

DOI: 10.1007/s10792-022-02418-y

PMC ID: 9902434

PubMed ID: 35932420

SO-VID: ffd413b1-28e1-4bfe-988f-dbbee639d42d

License:

Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

History

Date received : 22 April 2022

Date accepted : 28 June 2022

Funding

Funded by: FundRef http://dx.doi.org/10.13039/100000053, National Eye Institute;

Award ID: R01EY030283

Award Recipient : Victor L. Perez

Custom metadata

ScienceOpen disciplines: Ophthalmology & Optometry

Keywords: melt electrowriting,ocular surface,ophthalmology,tissue engineering

Data availability:

ScienceOpen disciplines: Ophthalmology & Optometry

Keywords: melt electrowriting, ocular surface, ophthalmology, tissue engineering

Establishment of a bi-layered tissue engineered conjunctiva using a 3D-printed melt electrowritten poly-(ε-caprolactone) scaffold

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Abstract

Purpose

Methods

Results

Conclusion

Supplementary Information

Related collections

Karger: Ophthalmology

Most cited references 43

The return of a forgotten polymer—Polycaprolactone in the 21st century

Direct writing by way of melt electrospinning.

Electrospun poly(epsilon-caprolactone) microfiber and multilayer nanofiber/microfiber scaffolds: characterization of scaffolds and measurement of cellular infiltration.

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