June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Generating cellular and collagen fibril alignment in RAFT tissue equivalents for corneal stroma reconstruction
Author Affiliations & Notes
  • Alvena K Kureshi
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Dev Mukhey
    Institute of Ophthalmology, University College London, London, United Kingdom
  • James B Phillips
    Biomaterials & Tissue Engineering, University College London, London, United Kingdom
  • James L Funderburgh
    University of Pittsburgh, Pittsburgh, PA
  • Julie T Daniels
    Institute of Ophthalmology, University College London, London, United Kingdom
  • Footnotes
    Commercial Relationships Alvena Kureshi, None; Dev Mukhey, None; James Phillips, None; James Funderburgh, None; Julie Daniels, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1948. doi:
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      Alvena K Kureshi, Dev Mukhey, James B Phillips, James L Funderburgh, Julie T Daniels; Generating cellular and collagen fibril alignment in RAFT tissue equivalents for corneal stroma reconstruction. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1948.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: The corneal stroma consists of a weave-like arrangement of collagen lamellae which facilitates transparency of the cornea. Currently RAFT (Real Architecture for 3D Tissue) equivalents have a random arrangement of cells and collagen fibrils. The aim of this work is to investigate the potential of corneal stromal stem cells (CSSCs) in generating cell and collagen fibril alignment in RAFT TE.

Methods: Free-floating collagen gels were used to establish an optimum cell seeding density and culture time for RAFT TE. CSSCs were seeded in tethered collagen gels and cultured for 8, 24 or 72 hours at 37°C to induce cell and fibril alignment. Cells generate forces contracting the tethered gel and align in the direction of principal strain in the middle (M) and edge (E) regions. An unaligned control region (delta)(D) exists within the gel where cells are stress-shielded. Gels were stabilized with RAFT process to retain cell and fibril alignment in the absence of tethering. CSSC markers (PAX6 and CD90) were used to confirm CSSC phenotype. Cell alignment was quantified with 3D image analysis. SEM and reflection confocal microscopy were used to assess fibril alignment.

Results: Free-floating collagen gels exhibited an average contraction of 65 ± 1.3% at 8 hours with 100,000 cells/gel (n=6). A high proportion of CSSCs had positive CD90 (89.9%) and PAX6 (98%) expression at the start of experiments. Following tethering (8hrs), CSSCs lost PAX6 expression in all regions of the gel, with mean percentage of cells expressing PAX6 in D (4.9 ± 0.9%), M (6.2 ± 0.7%) and E (4.8 ± 1.1%). Cell and fibril alignment was observed in the direction of principal strain in M and E regions of RAFT, but absent in D region. At 8 hours, mean angle of cell alignment was significantly greater for M and E regions at 24° and 12° respectively, compared to 48° in D region (p<0.0001, n=3), with a greater difference following 24 and 72 hours culture (n=1).

Conclusions: Alignment of CSSCs was associated with fibril alignment in tethered RAFT TE, indicating CSSCs may play an organizational role in the developing stroma. Loss of PAX6 expression following tethering suggests CSSCs may differentiate into keratocytes. Tethered RAFT TE shared structural resemblance with stromal lamellae and may form the basis of a corneal stromal tissue equivalent. This model could aid further studies into which mechanisms regulate CSSC differentiation.

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