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Praveena Gupta, Stephanie Vega, Joan Nichols, Joachim Cortiella, Massoud Motamedi, Bernard F. Godley, Gracie Vargas; Characterization of Decellularized Cornea Using Nonlinear Optical Microscopy. Invest. Ophthalmol. Vis. Sci. 2012;53(14):284. doi: https://doi.org/.
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Recent advances in tissue engineering have led to the development of decellularized corneal matrix as a promising native scaffold in engineered transplantable corneas. There is a need for noninvasive spatial and temporal monitoring of quality and integrity of the tissue matrix and cultures toward optimizing the design of transplantable corneas. The aim of this study was to characterize and monitor the subcellular depth-resolved properties of the decellurized cornea for tissue engineering by noninvasive imaging using multimodal nonlinear microscopy, including multiphoton microscopy and second harmonic generation microscopy (SHG).
Freshly trephined pig corneas were processed by incubating in 2-4% sodium dodecyl sulfate and placed into a rotating bioreactor containing 1% SDS for 3-5 weeks at room temperature. Subsets of corneas were exposed to riboflavin/UVA cross linking prior to processing for decellularization. Unstained decellularized corneas were imaged by multiphoton autofluorescence microscopy (MPAM) and SHG to evaluate the matrix architecture, followed by histology to visualize the different layer of the cornea. To confirm that the acellular matrix is biocompatible, decellularized corneas were incubated with limbal stem cells at different time points and then imaged for cell survivability and growth. Cells were stained with Hoechst 33342 and Calcein-AM Green to visualize the nucleus and cytoplasm of live cells respectively by two-photon microscopy.
Porcine corneas were decellularized completely as evidenced by autofluorescence/Hoechst imaging. The fibrillar collagen architecture was preserved after the decellularization process as analyzed by SHG. The UVA cross linking actually prevented the tissue from mechanical damage and decreased osmotic swelling in culture medium. The regular lamellar structure of the cornea was maintained along with its transparency even after three-four weeks of culture, especially in irradiated corneas. Sheets of epithelial cells were formed on the apical surface of the matrix after repopulating with corneal limbal stem cells in culture medium.
Multimodal nonlinear optical microscopy provided assessment to depths not achievable by traditional confocal microscopy and provided valuable information regarding matrix integrity and makeup as well as monitoring of the ex vivo culture. Engineered corneal bio-matrix may be an ideal scaffold to repair, repopulate scarred corneal tissue for transplantation purposes.
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