Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Summary

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Abstract

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Introduction

Nanostructured surfaces have recently attracted substantial attention due to their various potential applications, including patterning, cell culture, cleaning, and surface switching. For example, superhydrophobic surfaces inspired by the nanostructure of the lotus leaf and other responsive surfaces are capable of reacting to external stimuli^1-4.

The Langmuir film is one of the most widely studied polymer coatings. A Langmuir film is formed by dropping amphiphilic molecules onto an air-water interface^5-8. The film can then be transferred onto a solid surface by physical or chemical adsorption, and the molecular conform....

Protocol

1. Synthesis of Polystyrene-block-poly(N-isopropylacrylamide) by Two-step Reversible Addition-fragmentation Chain Transfer (RAFT) Radical Polymerization

1. Dissolve styrene (153.6 mmol), 4-cyano-4-(ethylsulfanylthiocarbonyl) sulfanylpentanoic acid (ECT; 0.2 mmol), and 4,4'-Azobis(4-cyanovaleric acid) (ACVA; 0.04 mmol) in 40 ml of 1,4-dioxane. Freeze the solution in liquid nitrogen under vacuum for 15-20 min to remove the reactive species and gradually thaw at RT. Make sure that the solution is completely thawed and repeat this freeze-pump-thaw degassing cycle three times.
2. Obtain the polystyrene (PSt) (Mw: 13,500) as a macro RAF....

Results

Block copolymers composed of polystyrene and poly(N-isopropylacrylamide) (St-IPAAms) with specific molecular weights were synthesized by RAFT radical polymerization. ECT was prepared as a chain-transfer agent as described in Moad et al.²⁸. Two St-IPAAm molecules of different PIPAAm chain lengths were synthesized, and the obtained block polymers were characterized by ¹H nuclear magnetic resonance (NMR) and gel permeation chromatography (GPC). The mol.......

Discussion

A temperature-responsive surface was fabricated by the Langmuir-Schaefer method, and the surface properties for cell adhesion/detachment and cell sheet recovery were optimized. When using this method for the fabrication of surfaces, several steps are critical. The molecular composition of the St-IPAAm molecules has a great effect on the surface structure and the stability of the surface, and by extension, on cell adhesion and detachment. In particular, the St-IPAAm molecules should have a narrow molecular weight distribu.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
N-isopropylacrylamide	Kohjin		No catalog number
Azobis(4-cyanovaleric acid)	Wako Pure Chemicals	016-19332
Styrene	Sigma-Aldrich	S4972
1,3,5-trioxane	Sigma-Aldrich	T81108
1,4-Dioxane	Wako Pure Chemicals	045-24491
DMEM	Sigma	D6429
PBS	Nakarai	11482-15
Streptomycin	GIBCO BRL	15140-163
Penicillin	GIBCO BRL	15140-122
Trypsin-EDTA	Sigma	T4174
FBS	Japan Bioserum	JBS-11501
BAECs	Health Science Reserch Resources Bank	JCRB0099
Cover Glasses	Matsunami Glass Industry	C024501
AFM NanoScope V	Veeco
1H NMR INOVA 400	Varian, Palo Alto
ATR/FT-IR NICOLET 6700	Thermo Scientific
GPC HLC-8320GPC	Tosoh
TSKgel Super AW2500, AW3000, AW4000	Tosoh
Langmuir-Blodgett Deposition Troughs	KSV Instruments	KN 2002	KSV NIWA Midium trough
Nikon ECLIPSE TE2000-U	Nikon