Micro- and nanotopography with extracellular matrix coating modulate human corneal endothelial cell behavior.
The human corneal endothelium plays an important role in maintaining corneal transparency. Human corneal endothelial cells have limited regenerative capability in vivo. Consequently, endothelial dysfunction can occur following corneal endothelial trauma or inherited diseases. To restore endothelial function, corneal transplantation is needed. However, there is a worldwide shortage of donor corneas, motivating the development of a tissue-engineered graft alternative using cultivated endothelial cells. To induce in vitro cell proliferation, much effort has been made to improve culture conditions and to mimic the native extracellular microenvironment. We incorporated topographical and biochemical cues in our in vitro culture of human corneal endothelial cell line B4G12 (HCEC-B4G12) and hypothesized that manipulation of the extracellular environment can modulate cell proliferation, morphometry and phenotype. The topographies tested were nanopillars, microwells and micropillars on polydimethylsiloxane, while the biochemical factors were extracellular matrix protein coatings of fibronectin-collagen I (FC), FNC® coating mix (FNC) and laminin-chondroitin sulfate (LC). Cellular morphometry, Na(+)/K(+)-ATPase and zona occludens 1 (ZO-1) gene and protein expression were analyzed 3days after cells had formed a confluent monolayer. The cell circularity on all patterns and coatings was above 0.78. On all coatings, cell area was the lowest on micropillars. The coefficient of variation (CV) of the cell area was the lowest on nanopillars with an LC coating. With an FC coating, micropillars induced a better cellular outcome as the cells had the greatest circularity, smallest cell area and highest Na(+)/K(+)-ATPase and ZO-1 gene and protein expression. With the LC coating, HCECs grown on nanopillars resulted in the lowest CV of the cell area and the highest ZO-1 gene expression. Thus, HCEC-B4G12 morphometry and phenotype can be improved using different topographical and biochemical cues.
Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore; Department of Biomedical Engineering, National University of Singapore, Singapore 117575, Singapore.
Stephanie
S
Koo
Department of Biomedical Engineering, National University of Singapore, Singapore 117575, Singapore; Institute of Materials Research and Engineering, A(∗)STAR (Agency for Science, Technology and Research), Singapore 117602, Singapore.
Rizwan
R
Muhammad
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 168751, Singapore.
Gary S L
GS
Peh
Tissue Engineering and Stem Cell Group, Singapore Eye Research Institute, Singapore 168751, Singapore; Singapore National Eye Centre, Singapore 168751, Singapore; Duke-NUS Graduate Medical School, Singapore 169857, Singapore.
Jodhbir S
JS
Mehta
Mechanobiology Institute, National University of Singapore, Singapore 117411, Singapore; Department of Biomedical Engineering, National University of Singapore, Singapore 117575, Singapore; Department of Surgery, National University of Singapore, Singapore 119228, Singapore. Electronic address: eyim@nus.edu.sg.
Evelyn K F
EK
Yim
D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D020099:Coated Materials, Biocompatible / Q000494:pharmacology*; D004129:Dimethylpolysiloxanes / Q000737:chemistry; D042783:Endothelial Cells / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D004728:Endothelium, Corneal / Q000166:cytology*; D005109:Extracellular Matrix / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D053758:Nanoparticles / Q000737:chemistry* / Q000648:ultrastructure; D060888:Real-Time Polymerase Chain Reaction; D000254:Sodium-Potassium-Exchanging ATPase / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling; D062826:Zonula Occludens-1 Protein / Q000235:genetics / Q000378:metabolism
D020099:Coated Materials, Biocompatible; D004129:Dimethylpolysiloxanes; D062826:Zonula Occludens-1 Protein; D000254:Sodium-Potassium-Exchanging ATPase; D001973:Bromodeoxyuridine
Cell proliferation; Extracellular matrix protein; HCEC-B4G12; Substrate topography; Tissue engineering