title: Decellularization of porcine corneas and repopulation with human corneal cells for tissue-engineered xenografts.
abstract: OBJECTIVE
  To evaluate the potential use of decellularized porcine corneas (DPCs) as a carrier matrix for cultivating human corneal cells in tissue engineering.
  
  
  METHODS
  Corneal cells were isolated from human corneoscleral rims. Porcine corneas were decellularized using hypotonic tris buffer, ethylene diamine tetra-acetic acid (EDTA, 0.1%), aprotinin (10 KIU/ml) and 0.3% sodium dodecyl sulphate. Haematoxylin-eosin (HE) and 4,6-diamidino-2-phenylindole (DAPI) staining were performed to confirm removal of the corneal cells. Quantitative analysis was performed to determine levels of desoxyribonucleic acid (DNA) using DNA Purification Kit (Fermentas, St. Leon-Rot, Germany). Alcian blue staining was carried out to analyse the structure of the extracellular matrix (ECM). Corneal stromal cells were injected into the DPCs; limbal corneal epithelial cells and corneal endothelial cells were seeded onto the anterior and posterior surfaces of the DPCs, respectively. Evaluation was undertaken at days 14 and 30. The phenotypical properties of the cultivated corneal cells were investigated using Immunolocalization of type I collagen, keratocan, lumican, cytokeratin 3 (AE5) and type VIII collagen.
  
  
  RESULTS
  Haematoxylin-eosin and DAPI staining showed efficient elimination of porcine corneal cells, whereas alcian blue confirmed gross preservation of the ECM. The quantitative analysis of the DNA content showed a significant reduction (mean before decellularization: 75.45 ± 13.71 ng/mg; mean after decellularization: 9.87 ± 2.04 ng/mg, p < 0.001). All three types of corneal cells were efficiently cultured and expanded on the DPCs.
  
  
  CONCLUSIONS
  Decellularized porcine corneas might serve as a potential scaffold for tissue engineering of the cornea, possibly providing xenogenic substrate for corneal transplantation.
authors.affiliation: Department of Ophthalmology, Eberhard-Karls University, 72076 Tuebingen, Germany. efdal.yoeruek@med.uni-tuebingen.de
authors.forename: Efdal
authors.initials: E
authors.lastname: Yoeruek
authors.forename: Tarek
authors.initials: T
authors.lastname: Bayyoud
authors.forename: Christine
authors.initials: C
authors.lastname: Maurus
authors.forename: Johanna
authors.initials: J
authors.lastname: Hofmann
authors.forename: Martin S
authors.initials: MS
authors.lastname: Spitzer
authors.forename: Karl-Ulrich
authors.initials: KU
authors.lastname: Bartz-Schmidt
authors.forename: Peter
authors.initials: P
authors.lastname: Szurman
mesh_terms: D000368:Aged; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D018929:Cell Culture Techniques; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D024042:Collagen Type I / Q000378:metabolism; D024102:Collagen Type VIII / Q000378:metabolism; D003315:Cornea; D060527:Corneal Keratocytes / Q000166:cytology* / Q000378:metabolism; D016039:Corneal Transplantation; D004247:DNA / Q000032:analysis; D016172:DNA Fingerprinting; D004728:Endothelium, Corneal / Q000166:cytology* / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology* / Q000378:metabolism; D005109:Extracellular Matrix; D006801:Humans; D007124:Immunoenzyme Techniques; D007632:Keratan Sulfate / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D000071234:Lumican; D009924:Organ Culture Techniques; D011509:Proteoglycans / Q000378:metabolism; D013552:Swine; D023822:Tissue Engineering; D054457:Tissue Scaffolds; D014183:Transplantation, Heterologous
chemical_list: D015415:Biomarkers; D011508:Chondroitin Sulfate Proteoglycans; D024042:Collagen Type I; D024102:Collagen Type VIII; C490948:KERA protein, human; D053558:Keratin-3; C000604306:LUM protein, human; D000071234:Lumican; D011509:Proteoglycans; D004247:DNA; D007632:Keratan Sulfate