Cell adhesion and accelerated detachment on the surface of temperature-sensitive chitosan and poly(N-isopropylacrylamide) hydrogels
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- Wang, J., Chen, L., Zhao, Y. et al. J Mater Sci: Mater Med (2009) 20: 583. doi:10.1007/s10856-008-3593-0
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A series of temperature-sensitive poly(NIPAAm-co-CSA) hydrogels were synthesized by the copolymerization of acrylic acid-derivatized Chitosan (CSA) and N-isopropylacrylamide (NIPAAm) in aqueous solution. Their swelling properties and L929 cell adhesion and detachment behaviors were studied. It was found that poly(NIPAAm-co-CSA) hydrogels were temperature-sensitive associated with the roles of the component PNIPAAm. Most significantly, poly(NIPAAm-co-CSA) hydrogels exhibited simultaneously good swelling properties. The investigation of L929 cell adhesion and detachment of poly(NIPAAm-co-CSA) hydrogels indicated the cell adhesion and spreading was higher on the surface of poly(NIPAAm-co-CSA) hydrogels than that of PNIPAAm hydrogel at 37°C due to the incorporation of CS, which having excellent cell affinity. Poly(NIPAAm-co-CSA) hydrogels showed more rapid detachment of cell sheet compared to PNIPAAm hydrogel because of the highly hydrophilic and hygroscopic nature of CS chains when reducing the culture temperature from 37°C to 20°C.
Nowadays, a polymer-grafted culture surface with temperature-sensitivity has been utilized for non-invasive recovery of several distinct to culture cell monolayers by manipulating the culture temperature without enzyme or chelator addition [1, 2]. Among the many intelligent polymers, poly(N-isopropylacrylamide) (PNIPAAm) is exploited for these applications because it exhibits coil-to-glouble changes with temperature in aqueous solution across its lower critical solution temperature (LCST) of 32°C [3, 4]. PNIPAAm is fully hydrated with an extended chain conformation in aqueous solution below 32°C and is extensively dehydrated and compact above 32°C. The LCST of NIPAAm copolymers can be controlled over a broad temperature range by adding copolymer compositions, making these polymers very interesting for medical and biotechnological applications. Due to unique thermal property, PNIPAAm and its copolymers have been widely studied in the fields of separation [5, 6], drug delivery [7–9], cell and tissue engineering [10, 11], and other switching devices .
Okano et al. has introduced PNIPAAm molecules onto solid surfaces to induce temperature-responsive hydrophilic/hydrophobic surface property alterations for several different applications [13–15]. On PNIPAAm-grafted surfaces, various cell types adhere and proliferate at 37°C. Upon reducing the culture temperature to 20°C, culture surfaces become hydrophilic, and cells adhered spontaneously detach along with their deposited extracellular matrix [16–18]. However, PNIPAAm is not good biocompatible, cell attach is restricted. Therefore, its biocompatility should be improved by fabricating PNIPAAm copolymers. Furthermore, spontaneous cell sheet detachment from surfaces of NIPAAm-grafted TCPS is relatively a slow process, occurring gradually from the sheet periphery toward the interior. Thus, significant incubation time at reduced temperature is required to lift up an intact cell sheet completely. Rapid detachment of cultured cell sheets is a very important recovery method that permits facile manipulation of the sheets. The rate-limiting step to cell sheet recovery is the hydration of the underlying PNIPAAm molecules grafted on the surface. Incorporation of a highly water permeable substrate to interface between the cell sheets and the thermo responsive polymer surfaces could accelerate the hydration of the hydrophobized PNIPAAm segments interacting with the cell sheet.
Chitosan (CS) is a positively charged specific polysaccharide, which stimulates cell growth and protein adsorption. It was reported that, besides its good biocompatility, CS has an excellent cell affinity [19, 20], and has good cellular proliferation and attachment properties in chondrogenic human mesenchymal stem cells (MSC). On the other hand, the hydrogels with porous conformation could accelerate the hydration of the material. In the present study, CS was introduced into PNIPAAm gel and the copolymerized hydrogels poly(NIPAAm-co-CSA) were prepared by the free radical copolymerization of NIPAAm and macromer CSA to achieve a much more cell attachment and growth, also a much more rapid cell sheet detachment in comparison with only PNIPAAm hydrogel.
2 Materials and methods
NIPAAm was purchased from Kohjin Co. Ltd., Japan and used after recrystallization from n-hexane. Chitosan (CS, Mr = 50,000, percentage of deacetylation degree: 85%) was provided by Shanghai Concachem Co., Ltd. Acrylic acid was obtained from Tianjin Chemical Reagent and purified by distillation. TCPS, Trypsin−EDTA solution, streptomycin, penicillin, and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Shanghai Sangon Biological Engineering Technology & Sevices Co., Ltd. All other reagents, including 1-ethyl-3-(3-dimethyl-aminopropyl)-carboiimide (EDC), peroxydisulfate (APS), N,N,N′,N′-tetra-methylenediamine (TEMED), N,N′-methylenebis(acrylamide) (MBAA), ethanol, etc., were of analytic grade and made in China, and were used as received without further purification.
2.2 Synthesis of CSA
To obtain a CS-based reactive monomer that can be copolymerized with NIPAAm to form a hydrogel, macromer CSA was synthesized. 1 g CS was dissolved in 100 ml hydrochloric acid (HCl) (1 wt.%) followed by the addition of 0.5 g of EDC and 0.1 ml TEMED, 15 ml acrylic acid was then added to the solution. The reaction mixture was stirred for 30 h at room temperature. After neutralization of acrylic acid and HCl with NaOH, the solution was purged by distilled water with stirring. The precipitate was collected by filtration and washed twice with ethanol for purification. The macromer CSA was dried under vacuum at room temperature for 24 h.
2.3 Synthesis of poly(NIPAAm-co-CSA) hydrogels
Poly(NIPAAm-co-CSA) hydrogels were prepared by the free radical copolymerization of NIPAAm and CSA in HCl solution using APS and TEMED as an initiating system. Specifically, CSA and NIPAAm were dissolved according to a weight ratio r, in the feed (NIPAAm/CSA) of 3, 4, 5, 6, and 7 respectively. APS (0.2 wt.%) was then added. After bubbling with nitrogen to remove oxygen, TEMED (0.5 wt.%) was added with stirring and the copolymerization proceeded at room temperature for 24 h. As a comparison, pure PNIPAAm hydrogel was prepared in the presence of MBAA (2 wt.%). The obtained hydrogels were cut into thin disks of 24 mm in diameter, and then immersed in distilled water for 1 week to remove the unreacted monomer.
CS, CSA and Poly(NIPAAm-co-CSA) hydrogels were powdered with KBr and pressed into pellets under reduced pressure to determine infrared spectra (Vector 22 FTIR, BRUKER Co., Germany). The same sample was analyzed by XPS (Perkin Elmer 5600 ESCA system).
The hydrogels were immersed in distilled water to swell for 24 h over a range of temperatures from 20°C to 50°C. The equilibrated swelling ratio (SR) was defined as Ws/Wd, where Ws and Wd were the weights of the swollen and dry gels, respectively.
To study the reswelling kinetics, hydrogels after equilibrium swelling at 37°C were immersed into distilled water of 20°C quickly. The hydrogels were weighed at different times. The reswelling ratio (RSR) was calculated by (Wt − W0)/Wd, where W0 and Wt were the weight of swollen gel at initial and t times, respectively.
The SR and RSR in cell culture medium were obtained as in water.
2.5 Cell culture on poly(NIPAAm-co-CSA) hydrogels
The ability of poly(NIPAAm-co-CSA) hydrogels to support cell attachment was tested using L929 cells in 12 well plates. The L929 cells were provided by Shanghai Queen & King Biochem Co,.LTD, and a cell bank was first created by expanding the cells on 25 cm2 flasks in a humidified environment of 95/5% air/CO2, and freezed the cells at 7.0 × 104 cells/ml (1 ml) in cryogenic vials. Cell freezing was achieved at a rate of 1°C/min and the cells were stored in liquid N2 until use.
For each experiment, a vial of frozen cells was thawed and seeded on 25 cm2 flasks. The cells were cultured in DMEM with 10% FBS and 1% penicillin/streptomycin until they reached about 80% confluence, and trypsinized once more for seeding onto polymer surfaces. The trypsinized cells were suspended in tissue culture medium and were diluted, then 2 ml of cell suspension was added to the hydrogels and TCPS to give 1 × 104 cells/cm2. Cell studies were typically completed during the subsequent 8 days with culture medium changes every 2 or 3 days.
The cell morphology was observed by phase-contrast microscopy (OLYMPUS ckx31, Japan). For the opacity hydrogels, the cell morphology was observed by Scanning Electron Microscope (SEM) (Quanta 200, FEI Co.). Since the cell seeding onto hydrogel surfaces could not sustain lyophilization and low-temperature treatment, sample preparations of the cell–hydrogel complexes for SEM were modified as follows. The specimens were first rinsed with 37°C PBS buffer and fixed in 2.5% glutaraldehyde (in 0.1 M phosphate buffer) at 37°C. After fixation for 24 h, the samples were washed in PBS buffer (37°C, 10 min) three times. Finally, the specimens were dried using a critical point dryer in liquid carbon dioxide, sputter-coated with gold, and visualized by SEM.
The number of cells adhered to a specific surface was quantified using a hemacytometer. For this method, the excess culture media was drained and 0.5 ml of trypsin/EDTA was added to each well. After about 5 min, 1 ml of fresh culture medium was added, cells were counted manually from five different fields and averaged, giving the number of cells/ml.
The 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di- phenytetrazoliumromide (MTT) assay was used to quantify metabolic activity in each well. At the desired time point, the culture medium were drained and re-supplied with 1 ml of fresh culture medium. MTT dissolved in HBSS (5 mg/ml) was added to each well. After incubating at 37°C for 4 h, excess medium was removed. The cells were then dissolved in 1.2 ml DMSO and the OD value of each well was determined by Auto microplate reader (Σ960, Metertech Co. America) while the wavelength was selected at 490 nm.
2.6 Detachment of single L929 cell
Detachment of single L929 cell was achieved using low temperature treatment after incubation at 37°C for 24 h. L929 cells were plated on each surface at a density of 3 × 104 cells/cm2 and cultured for 24 h to allow attachment and spreading on each polymer surface. For low temperature treatment, the spread cells were transferred to a CO2 incubation equipped with a cooling unit fixed at 20°C. After 5, 10, 15, 20, 25, 30, 35, and 40 min incubation, the sing cell detached was counted using a hemacytometer.
2.7 Detachment of confluently cultured L929 cell sheets
L929 cells were plated onto each surface at a density of 5 × 104 and cultured at 37°C. After 24 h cultivation, unattached cells were removed by medium change. Cells were cultured for 6 days after reaching confluence, and each plate was transferred to the CO2 incubator equipped with a cooling unit fixed at 20°C, and periodically the cells detached were counted using a hemacytometer. All procedures were carefully carried out not to alter the incubator temperature because the cell culture surfaces used in this experiment were all thermo responsive.
3 Results and discussion
3.1 Characterization of CSA and poly(NIPAAm-co-CSA) hydrogel
The XPS analysis results showed the N composition of CSA (3.6) was lower than that of CS (4.8), C composition of CSA (72.4) was higher than that of CS (69.4) because of the introduction of AAc, indicating that the reaction between CS and AAc occurred. The degree of substitution of CSA (DS, the molar ratio of AAc per CS unit), determined by XPS was 2.2 in this study .
3.2 Thermo-responsive properties of hydrogels
3.3 Cell attachment and growth
3.4 Single cell detachment
Since poly(NIPAAm-co-CSA) hydrogels exhibit well-defined temperature sensitivity, it is expected that cells cultured on poly(NIPAAm-co-CSA) hydrogels could be detached simply by decreasing the temperature from 37°C (hydrophobic) to 20°C (hydrophilic). It was observed that when the culture temperature was reduced to 20°C after 24 h incubation at 37°C during which almost all of the seeded cells were attached and spread on those surfaces, the spread cells were rounded and detached from the surfaces.
3.5 Detachment of cell sheets
3.6 Cell transshipment
The characteristics of the detached cells on poly(NIPAAm-co-CSA) hydrogels (at 20°C) were examined by the total metabolic activity. The L929 cells detached from hydrogels and digested from TCPS were seeded respectively onto dishes at a cell density of 2 × 104 cells/cm2.
In this work, acrylic acid-derivatized Chitosan (CSA) and a series of poly(NIPAAm-co-CSA) hydrogels were successfully synthesized and their swelling behaviors were investigated. It was found that poly(NIPAAm-co-CSA) hydrogels were temperature-sensitive and exhibited much better swelling properties than the PNIPAAm gel.
A study of L929 cells cutured on the hydrogels indicated that the cell adhesion and spreading was higher on surfaces of poly(NIPAAm-co-CSA) hydrogels compared with PNIPAAm hydrogel, suggesting that the incorporation of CS were capable of enhancing the attachment of L929 cells. When the temperature decreased, the poly(NIPAAm-co-CSA) hydrogel showed hydrophilic and the cells spontaneously detached along with their deposited extracellular matrix. The existence of co-polymerized CS chains could accelerate cell detachment. This step protected the cell function well.
This research was financially supported by the National Nature Science Foundation of China (Contract grant number: 20574051), and the Specialized Research Fund for the Doctoral Program of Higher Education (Contract grant number: 20050058001).