title: Relationships between surface roughness/stiffness of chitosan coatings and fabrication of corneal keratocyte spheroids: Effect of degree of deacetylation.
abstract: Fabrication of the cell spheroids from corneal keratocytes has important implications to the advance in tissue engineering while stimulation from the interface of a biopolymer coating has the ability to modulate this event. This study aims to investigate the dependence of keratocyte migration, proliferation, and differentiation on the surface roughness/stiffness of the chitosan coatings through modifications by degree of deacetylation (DD). After a series of deacetylation process, chitosan coatings with increasing DD exhibited significantly decreased surface roughness and increased surface stiffness. Relationships between the behaviors of rabbit corneal keratocytes (RCKs) and biopolymer coatings with varying DDs (between 75% and 96%) were also found during in vitro cultivation. Both the surface roughness increase and stiffness decrease could lead to enhanced cell migration, which is the main driving force for the early stage spheroid formation on chitosan substrates (e.g., within 8h). With these stimulations from the substrate interfaces, the size and morphology of RCK spheroids were greatly affected by the DD of chitosan. When fabricated on a lowered DD of chitosan material, the spheroids had a larger size with abundant extracellular matrix produced around the cells. At a later stage of spheroid cultivation (e.g., 5 days), significantly higher amount of RCKs on chitosan coatings was noted with increasing DD, indicating the substrate interface effects on cell proliferation. The keratocan expression of RCK spheroids grown on a lowered DD of chitosan was up-regulated, suggesting that both the surface roughness increase and stiffness decrease may facilitate the microenvironment for preservation of cellular phenotype. Overall, our work contributes to the scientific understanding of the keratocyte behaviors and spheroid fabrications in response to DD-mediated surface roughness/stiffness of chitosan coatings.
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China; Department of Bioengineering, University of Washington, Seattle, WA 98195-5061, USA.
authors.forename: Shih-Feng
authors.initials: SF
authors.lastname: Chou
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China; Biomedical Engineering Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China; Molecular Medicine Research Center, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China; Center for Tissue Engineering, Chang Gung Memorial Hospital, Taoyuan, Taiwan 33305, Republic of China. Electronic address: jylai@mail.cgu.edu.tw.
authors.forename: Jui-Yang
authors.initials: JY
authors.lastname: Lai
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China.
authors.forename: Ching-Hsien
authors.initials: CH
authors.lastname: Cho
authors.affiliation: Institute of Biochemical and Biomedical Engineering, Chang Gung University, Taoyuan, Taiwan 33302, Republic of China.
authors.forename: Chih-Hung
authors.initials: CH
authors.lastname: Lee
mesh_terms: D000107:Acetylation; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation; D002465:Cell Movement / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048271:Chitosan / Q000737:chemistry / Q000494:pharmacology*; D020099:Coated Materials, Biocompatible / Q000737:chemistry / Q000494:pharmacology*; D060527:Corneal Keratocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D005109:Extracellular Matrix / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D061251:Primary Cell Culture; D011509:Proteoglycans / Q000378:metabolism; D011817:Rabbits; D018874:Spheroids, Cellular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D013499:Surface Properties
chemical_list: D015415:Biomarkers; D020099:Coated Materials, Biocompatible; D011509:Proteoglycans; D048271:Chitosan
keywords: Chitosan coating; Corneal keratocyte; Degree of deacetylation; Spheroid fabrication; Surface roughness/stiffness