Keratocyte behavior in three-dimensional photopolymerizable poly(ethylene glycol) hydrogels.
The goal of this study was to evaluate three-dimensional (3-D) poly(ethylene glycol) (PEG) hydrogels as a culture system for studying corneal keratocytes. Bovine keratocytes were subcultured in DMEM/F-12 containing 10% fetal bovine serum (FBS) through passage 5. Primary keratocytes (P0) and corneal fibroblasts from passages 1 (P1) and 3 (P3) were photoencapsulated at various cell concentrations in PEG hydrogels via brief exposure to light. Additional hydrogels contained adhesive YRGDS and nonadhesive YRDGS peptides. Hydrogel constructs were cultured in DMEM/F-12 with 10% FBS for 2 and 4 weeks. Cell viability was assessed by DNA quantification and vital staining. Biglycan, type I collagen, type III collagen, keratocan and lumican expression were determined by reverse transcriptase-polymerase chain reaction. Deposition of type I collagen, type III collagen and keratan sulfate (KS)-containing matrix components was visualized using confocal microscopy. Keratocytes in a monolayer lost their stellate morphology and keratocan expression, displayed elongated cell bodies, and up-regulated biglycan, type I collagen and type III collagen characteristic of corneal fibroblasts. Encapsulated keratocytes remained viable for 4 weeks with spherical morphologies. Hydrogels supported production of KS, type I collagen and type III collagen matrix components. PEG-based hydrogels can support keratocyte viability and matrix production. 3-D hydrogel culture can stabilize but not restore the keratocyte phenotype. This novel application of PEG hydrogels has potential use in the study of corneal keratocytes in a 3-D environment.
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.|Fundacion INASMET, San Sebastian, Spain.
Nerea
N
Garagorri
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
Sara
S
Fermanian
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
Richard
R
Thibault
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
Winnette McIntosh
WM
Ambrose
Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA.|School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
Oliver D
OD
Schein
Department of Ophthalmology, Johns Hopkins University, Baltimore, MD, USA.|School of Medicine, Johns Hopkins University, Baltimore, MD, USA.
Shukti
S
Chakravarti
Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD, USA.
Jennifer
J
Elisseeff
D000818:Animals; D058578:Biglycan; D001672:Biocompatible Materials / Q000737:chemistry*; D002417:Cattle; D018929:Cell Culture Techniques / Q000379:methods*; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D011508:Chondroitin Sulfate Proteoglycans / Q000378:metabolism; D003094:Collagen / Q000378:metabolism; D003315:Cornea / Q000166:cytology* / Q000378:metabolism*; D016326:Extracellular Matrix Proteins / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D020100:Hydrogels / Q000737:chemistry; D007632:Keratan Sulfate / Q000378:metabolism; D000071234:Lumican; D008422:Materials Testing; D010777:Photochemistry / Q000379:methods; D011092:Polyethylene Glycols / Q000737:chemistry*; D011509:Proteoglycans / Q000378:metabolism; D023822:Tissue Engineering / Q000379:methods*
D058578:Biglycan; D001672:Biocompatible Materials; D011508:Chondroitin Sulfate Proteoglycans; D016326:Extracellular Matrix Proteins; D020100:Hydrogels; D000071234:Lumican; D011509:Proteoglycans; D011092:Polyethylene Glycols; D003094:Collagen; D007632:Keratan Sulfate