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Martha L. Funderburgh, Xia Li, Mary M. Mann, James L. Funderburgh; Expression of Corneal Stromal Matrix by Adipose-Derived Stem Cells in Compressed Collagen Gels. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5150.
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In spite of corneal immune privilege, many penetrating keratoplasties fail due to rejection, prompting interest in bioengineering of cornea from autologous cells. We recently showed human adipose-derived stem cells (ADSC) to produce stroma-specific extracellular matrix components in vitro (Du et al. Mol Vis, 2010); however, these constructs lack the organization and strength required of stromal lamellar grafts. Recent studies have shown that compression of collagen gels markedly increases the strength and integrity of this material. Here we tested the potential for ADSC to produce stromal extracellular matrix when encapsulated in compressed collagen.
Human ADSC were prepared and characterized as previously described. ADSC were incorporated into gels of neutralized acid-soluble 1.5% rat tail collagen in PBS and gelled 4 hours at 37 °C. Gels were compacted by steel weights on a glass plate with gels supported by nylon and stainless mesh over absorbent paper. After 5’ compression, gels were cultured in Advanced DMEM with FGF2, ascorbate 2-phosphate, and antibiotics for 1-2 weeks. Media were analyzed by Western blot for secreted keratocan and keratan sulfate and cellular mRNA analyzed for expression of markers for keratocyte phenotype (CHST6, KERA, B3GNT7, AQP1) by qPCR. Fixed gels were immunostained to detect actin, collagen, keratan sulfate, and keratocan by confocal microscopy.
ADSC cultured in compressed collagen maintained viability throughout 2 weeks of culture and secreted keratocan and keratan sulfate, unique stromal matrix components. Expression of keratocyte markers was upregulated in compressed gels. Gels maintained integrity and transparency, even forming a multilayered tissue-like construct, suggesting promise for their use as intrastromal implants.
Encapsulation of ADSC in compressed collagen produces a construct with increased strength and transparency compared to pellet cultures and hydrated collagen gels. Keratocyte matrix expression by ADSC in these constructs suggests suitability for use as autologous lamellar grafts or as intracorneal implants for refractive purposes.
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