title: The mechanism for keratinocyte detaching from pH-responsive chitosan.
abstract: In this study, we compared the detachment ratio of HaCaT and Hs68 cells from pH-responsive chitosan surface by raising medium pH from 7.20 to 7.65 for 60 min. The detachment ratio of elongated Hs68 cells was over 75%, but that of round-shaped HaCaT cells was less than 50%, even extending the incubation time to 6 h or enhancing the cytoskeletal contractile force with the Rho activator CN01. However, the addition of 2 mm of EDTA into the medium at pH 7.65 could effectively detach HaCaT cells (detachment ratio > 90%), indicating that the calcium ion played an important role in the detachment process. Therefore, the family of Ca(+2)-dependent integrin receptors was examined by RT-PCR, real-time PCR and immunocytochemistry. It was found the expression of integrin β4 (ITGb4) was HaCaT cell-specific and the mRNA level of ITGb4 in undetached HaCaT cells was significantly higher than that in detached ones. By modulating ITGb4 activity with specific functional blocking antibody ASC-8, the detachment ratio of HaCaT cells could be increased to be greater than 85%. Conversely, the addition of the ligand of ITGb4 laminin into the culture system decreased the medium pH-induced detachment ratio for HaCaT cells, but not for Hs68 cells. Further addition of ASC-8 could rescue the effect of laminin on preventing the detachment of HaCaT cells from pH-sensitive chitosan surface. Therefore, this study demonstrated the interaction of ITGb4 and laminin played an important role in controlling the detachment of HaCaT cells on pH-responsive chitosan.
authors.affiliation: Institute of Polymer Science and Engineering, College of Engineering, National Taiwan University, Taipei, 106, Taiwan.
authors.forename: Yi-Hsin
authors.initials: YH
authors.lastname: Chen
authors.affiliation: Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, 100, Taiwan.
authors.forename: Shao-Hsuan
authors.initials: SH
authors.lastname: Chang
authors.affiliation: Department of Ophthalmology, National Taiwan University Hospital, Taipei, 100, Taiwan. Electronic address: ijong@ms8.hinet.net.
authors.forename: I-Jong
authors.initials: IJ
authors.lastname: Wang
authors.affiliation: Institute of Polymer Science and Engineering, College of Engineering, National Taiwan University, Taipei, 106, Taiwan; Institute of Biomedical Engineering, College of Medicine and College of Engineering, National Taiwan University, Taipei, 100, Taiwan. Electronic address: thyoung@ntu.edu.tw.
authors.forename: Tai-Horng
authors.initials: TH
authors.lastname: Young
mesh_terms: D002460:Cell Line; D048271:Chitosan / Q000737:chemistry*; D003470:Culture Media / Q000737:chemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007150:Immunohistochemistry; D039663:Integrin beta4 / Q000378:metabolism; D015603:Keratinocytes / Q000166:cytology*; D007797:Laminin / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D003470:Culture Media; C059834:ITGB4 protein, human; D039663:Integrin beta4; D007797:Laminin; D048271:Chitosan
keywords: HaCaT cells; Integrin β4 (ITGb4); Laminin; pH-responsive chitosan