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Jian Wu, Yiqin Du, William R. Wagner, James L. Funderburgh; Corneal Stromal Tissue Bioengineered by Human Corneal Stromal Stem Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5152.
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Adult human corneal stromal stem cells (hCSSC) adopt keratocyte phenotype and secrete extracellular matrix (ECM) components unique to corneal stroma. In order to produce ECM suitable for corneal reconstruction this study tested the hypothesis that aligned scaffold of biodegradable nanofibers can act as a template to initiate and guide organization of a human corneal stroma-like ECM by hCSSCs.
Aligned nanofibrous scaffolding was prepared by the electro-spinning process using biodegradable poly(ester urethane) urea (PEUU) and used for substratum for culture of hCSSCs in serum-free media for periods up to 6 weeks. ECM secreted by hCSSCs was characterized by scanning electron microscopy (SEM), transmission electron microscopy (TEM), second harmonic generated (SHG) microscopy, and light-level immunohistochemistry. Gene expression was examined by quantitative RT-PCR.
SEM and TEM micrographs showed the nanofibrous PEUU scaffold to induce rapid alignment of the hCSSCs. After 6 weeks, 8-10 microm ECM layers formed on top of the scaffolding consisting of aligned periodic banded fibrils (67 nm) of a uniform fibril diameter (40.2±2.7 nm) and spacing (60.9±6.5 nm). SHG and SEM images demonstrated global alignment of these collagen fibrils throughout the construct. PEUU scaffolding lacking aligned nanofiber structure did not generate ECM of a similar level of organization. Immunofluorescent staining of the aligned constructs showed ECM to be abundant in type-I collagen and also collagens and proteoglycans typifying the unique ECM of human corneal stromal tissue. Gene expression profile showed hCSSCs gradually lost gene expression of adult stem cells (i.e., ABCG2), and substantially upregulated expression of genes marking keratocyte phenotype such as KERA, ALDH, PTGDS and CHST6.
Aligned nanofibrous scaffold prepared from biodegradable PEUU induces hCSSC to secrete and organize collagen-based lamellae with oriented fibers of uniform fiber diameter and spacing remarkably similar to those of human corneal stroma. The physical microenvironment of the nanofiber scaffold contributed an organizational role independent from its chemical composition. The global nature of the ECM organization suggests its suitability for use in corneal bioengineering.
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