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Biomacromolecules 2008, 9, 426–430
Cell Adhesion and Proliferation on Poly(N-vinylacetamide) Hydrogels and Double Network Approaches for Changing Cellular Affinities Hiroharu Ajiro,†,‡ Junji Watanabe,† and Mitsuru Akashi*,†,‡ Department of Applied Chemistry, Graduate School of Engineering, Osaka University, 2-1 Yamada-oka, Suita, Osaka 565-0871, Japan, and The Center for Advanced Medical Engineering and Informatics, Osaka University, 2-2 Yamada-oka, Suita, Osaka, 565-0871, Japan Received November 6, 2007; Revised Manuscript Received December 14, 2007
Poly(N-vinylacetamide) hydrogels (PNVA gels) were synthesized to investigate their basic characteristics for biomedical applications such as water contact angles, protein uptake, and mouse fibroblasts (L-929) cell adhesion. Because PNVA gels show hydrophilic features, double network (DN) hydrogels were prepared by the secondary polymerization of N-vinylacetamide (NVA) or acrylamide (AAm) in PNVA gels (NVA/NVA DN gels and NVA/ AAm DN gels, respectively), in order to vary PNVA gel features for biocompatibility. Contact angles for both DN gels decreased to around 20°, whereas both PNVA and PAAm gels were over 30°. On the other hand, more protein tended to adsorb to DN gels than single network hydrogels. Compared to PNVA gel, cell adhesion and proliferation on NVA/NVA DN gel were improved with less swelling ratio and much protein uptake, while no significant difference was observed on NVA/AAm DN gel, probably due to more hydrophilic character, supported by lowest water contact angle. These complicated structure change in DN gels would provide a new methodology for tuning the biocompatibility of hydrogels and for controlling surface hydrophilic characteristics and network structures.
Introduction Hydrogels are composed of large amounts of water and small amounts of polymers. The aforementioned characteristic makes them suitable for biomedical applications, including contact lenses and for coating blood contacting materials. Numerous studies on hydrogels for producing novel biomaterials have been recently reported such as tissue engineering scaffolds,1–4 3Dengineered tissues,5,6 drug delivery systems,7,8 and transdermal therapeutic systems (TTS).9 Because contacts with living bodies are required for all the aforementioned applications, suitable chemical structures and polymer component bioaffinities in hydrogels are important, even when only a small amount of polymer chains is present on hydrogel surfaces. Therefore, the design of chemical structures for monomers or comonomers,10 swelling ratios, and polymerization conditions is indispensable. For decades, our research group has investigated N-vinylamide polymers and hydrogels. These monomers bear vinyl groups on nitrogen atoms and various alkyl side chains bound by amide linkages. Poly(N-vinylalkylamide)s and poly(N-vinylamine) biofunctions have been studied such as lower critical solution temperatures, and thermal and pH responses.11–14 Because of their structural similarity to poly(acrylamide) derivatives and polypeptides, similar biofunctions as biomacromolecules are expected. Among the N-vinylamides derivatives, N-vinylacetamide (NVA) has drawn attention as an amphiphilic and aprotic vinyl monomer,15,16 and is considered to have potential applications for biomaterials. For example, NVA was used for coating on * Corresponding author. E-mail:
[email protected]. Telephone: +81-6-6879-7356. Fax: +81-6879-7359. † Department of Applied Chemistry, Graduate School of Engineering, Osaka University. ‡ The Center for Advanced Medical Engineering and Informatics, Osaka University.
polyethylene film by graft polymerization to improve their surface hydrophilicity.17 Nanoparticles prepared with NVA have the unique characteristic that salmon calcitonin (sCT) does not adsorb, whereas poly(methacrylic acid), poly(vinylamine), and poly(N-isopropylacrylamide) capture sCT protein molecules in rat gastrointestinal tracts.18,19 Recently, poly(N-vinylacetamide) hydrogels (PNVA gels) were used as patches with respect to TTS.20,21 However, other biomedical applications of PNVA have not been reported at the present time. In general, with the exception of replacing substituents, a monomer ratio change in copolymers is one of the best strategies for tuning material characteristics. For instance, an increase in the amount of phosphonic acid to around 40% in poly(vinyl phosphonic acid-co-acrylamide) hydrogels results in improved cell adhesion.22 One of the reasons for the limited application of PNVA is its poor copolymerization ability with other vinyl monomers due to the nonconjugated NVA structures between carbonyl and vinyl groups, which cause lower radical reactivities on vinyl groups.15 This different radical polymerizability cannot produce varied combinations with other vinyl monomers for tuning supplemental functionalities. To add to this, in order to properly introduce cross-linking points in hydrogels, crosslinkers with NVA analogue structures are required for equi reactivities such as N,N-methylene-bis-N-vinylacetamide (Mbis-NVA).23 Thus, an alternative strategy has been sought for improving components and the reliable creation of cross-linking points. On the other hand, Gong and Osada invented a methodology to increase the mechanical strength of hydrogels by a double network (DN) structure,24 whereas Mooney et al. strengthened hydrogels by introducing suitable cross-linkers.25 DN gels are a kind of interpenetrated (IPN) gel prepared during a two-step gel formation process. It is noteworthy that DN gels are composed of totally different monomers regardless of reactivity,
10.1021/bm701221c CCC: $40.75 2008 American Chemical Society Published on Web 01/08/2008
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Table 1. Hydrogel Preparationa first network entry
sample
monomer
cross-linker
1 2 3 4
PAAM gel PNVA gel NVA/NVA DN gel NVA/AAM DN gel
AAm NVA NVA NVA
MBAAm 5ON-bis-NVA 5ON-bis-NVA 5ON-bis-NVA
second network monomer
NVA AAm
cross-linker
thicknessb/mm
swelling ratioc
5ON-bis-NVA MBAAm
1.0 1.7 2.2 2.0
9.1 14.4 9.9 9.0
a [monomer]:[cross-linker]:[initiator] ) 100:1:1, [monomer] ) 2.0 mol/L. Solvent ) water, initiator ) 2,2′-azobis(2-methylpropionamidine) dihydrochloride (V-50). Monomer solution or first network hydrogel were placed between double glass plates that were separated by a 1 mm silicon gasket. b Gel membrane thickness after swollen. c Swelling ratio was defined as (Ws - Wd)/Wd.
which permits the preparation of gels with different monomers, even if they do not form chemical bonds by reacting directly with one another. Therefore, DN creation seems to be the perfect approach for improving and diversifying PNVA gels. As well as mechanical strength, a difference in biomaterial functionality is also important between single network (SN) PNVA gels and DN hydrogels, such that NVA was secondarily polymerized in the PNVA gel (NVA/NVA DN gel). With those interests, we recently reported a NVA/NVA DN gel (1.6 MPa) to compensate for the poor copolymerization ability of NVA (0.51 MPa), which resulted in a modest mechanical improvement.26 In this communication, we carried out a basic biocompatibility characterization on both PNVA and NVA/NVA DN gels by using a DN hydrogel with acrylamide (AAm) that was secondarily polymerized in PNVA gels (NVA/AAm DN gels) for comparison. We discovered that cell adhesion improved on NVA/NVA DN gels, and this anomalous phenomenon is discussed together with water contact angles and protein uptake to hydrogels.
Experimental Section Materials. NVA (Showa Denko K.K., Japan) was recrystallized from toluene/cyclohexane (1:3) and dried in vacuum at room temperature. AAm (Tokyo Chemical Industry Co., Ltd., Japan) and N,N-methylenebisacrylamide (MBAAm) (Tokyo Chemical Industry Co., Ltd., Japan) were recrystallized from acetone and dried in vacuum at room temperature. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (V50) (Wako, Japan) was used without further purification. N,N-5oxanonamethyene-bis-N-vinylacetamide (5ON-bis-NVA) was used as a NVA cross-linker and was prepared according to a previously reported method.27 Polymerization. A typical procedure for PNVA gels is as follows: NVA (0.34 g, 4 mmol), V-50 as a radical initiator (11 mg, 0.04 mmol), and each cross-linker (0.04 mmol) were dissolved in degassed water (2 mL). Solutions were injected between double glass plates that were separated by a silicon gasket (1.0 mm thickness) under a nitrogen atmosphere. The mixture was then polymerized at 37 °C for 4 h. The polymerization proceeded quantitatively with each first preparation (>99% yield). Gel membranes were cut into disks (9 and 6 mm diameters). The obtained SN gels were immersed in a second aqueous NVA or AAm solution (2 mol/L) containing 1 mol % cross-linker and 1 mol % V-50 at 4 °C for 1 day until equilibrium was reached. A second network was synthesized by heating at 37 °C for 6 h between double glass plates (1.0 mm thickness). Each hydrogel was immersed into a large amount of distilled water for 1 week to remove reaction residues. Yields were determined by increased weight: >99% for NVA/ AAm DN gels and 86% for NVA/NVA DN gels. The swelling ratio of the hydrogel was calculated by the following equation: (Ws - Wd)/ Wd, where Ws is the weight of the swollen hydrogel at room temperature and Wd is that of the dried gel. Contact Angles. Static contact angles were measured using an automatic contact angle meter apparatus (Drop Master 100, Kyowa Interface Science, Co. Ltd., Japan) at room temperature. A drop of ultrapure water was introduced onto the gel using a microsyringe.
Protein Uptake. Protein uptake was examined using bovine serum albumin (BSA), bovine gamma globulin (BγG), and bovine plasma fibrinogen (BPF) (Sigma, St. Louis, MO). Protein concentrations were adjusted to 4.5, 1.0, and 0.3 mg/mL in phosphate buffer saline (PBS). Hydrogels were cut into disks (6 mm diameters) and swollen with ultrapure water. They were then incubated in each protein solution (900 µL) (pH 7.4) for 4 h. After rinsing with PBS, adsorbed proteins were removed by 1 wt % of n-sodium dodecyl sulfate (SDS) (1000 µL) for 4 h. Proteins recovered from hydrogels were evaluated by a Micro BCA kit (Pierce, Rockford, IL). The absorbed protein per hydrogel, W (µg/unit) was defined as:
W ) W0 × {(VBCA + V) ⁄ V} × (VSDS ⁄ V)
(1)
where VBCA is the volume of the BCA solution (400 µL), V is the volume of the SDS solution with protein for measurement (400 µL), and VSDS is the volume of the SDS solution while removing proteins from the hydrogel (1000 µL). W0 (µg/mL) is calculated by the absorbance at 560 nm using a Micro BCA kit. Then, adsorbed protein per area and volume, which represents W1 (µg/cm2) and W2 (µg/mL), respectively, were calculated as below.
W1 ) W ⁄ (2 × πr2 + 2πr × d)
(2)
W2 ) W ⁄ (πr2 × d)
(3)
where r is the radius of each gel (3 mm) and d is the thickness of the hydrogel after swelling (Table 1). Cell Adhesion and Proliferation. Mouse fibroblast (L-929) cells were purchased from the RIKEN cell bank (Saitama, Japan) and routinely cultured in Eagle’s minimum essential medium (E-MEM, Nissui, Tokyo, Japan) at 37 °C under a 5% CO2 atmosphere. After treatment with 0.25% trypsin, the cell density was adjusted to 1.0 × 106 cells/mL, and 5.0 × 104 cells were seeded onto each hydrogel disk (9 mm diameter). Cells were cultured for 8 days, and the E-MEM was changed every other day. Hydrogels were transferred to another well for rinsing with 700 µL of PBS solution and were dipped into a 200 µL aliquot of a 0.25% trypsin solution for 1 min. Samples were combined with 500 µL of fresh E-MEM, and cells were collected by centrifugation. Cell pellets were dispersed with a 200 µL aliquot of a E-MEM solution, and 1/10 of the aliquot was removed and mixed with 20 µL trypan blue to count the number of cells using a hemocytometer.
Results and Discussion Structures of monomers and cross-linkers are shown in Figure 1. It is known that the NVA cross-linker itself reacts intermolecularly to produce cyclic molecules when two cross-linker vinyl groups are short, such as M-bis-NVA.28 To avoid side reactions and to keep the hydrophilicity in the long alkyl chain between two vinyl groups, we employed the cross-linker, 5ONbis-NVA.26 In this study, we synthesized four kinds of hydrogels: a PNVA gel (Table 1, entry 2), a NVA/NVA DN gel (Table 1, entry 3), and a NVA/AAm DN gel (Table 1, entry 4), using a poly(acrylamide) hydrogel (PAAm gel) as a control (Table 1 entry 1) because of the chemical structure similarity. Both DN gel
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Figure 1. Monomer structures and schematic illustration of hydrogel preparation.
Figure 2. Static contact angle on the PAAm gel (a), PNVA gel (b), NVA/NVA DN gel (c), and NVA/AAm DN gel.
swelling ratios decreased, implying that a secondary network had developed inside PNVA gels (Table 1, entries 3 and 4). PAAm, PNVA, and NVA/NVA DN gels (Table 1, entries 1, 2, and 3) were quite transparent, but NVA/AAm DN gels were slightly cloudy (Table 1, entry 4), although the hydrogel was obtained quantitatively. This implies that the second monomer NVA spread homogeneously in the first network, whereas the AAm might be partially crystallized during polymerization, as it is a different monomer from the first PNVA gel (photos are available in the Supporting Information, Figure S1). In general, the water contact angle is an indication of cell adhesion. In other words, the most favorable protein adsorption and cell adhesion on the polymer surface occurs when the contact angle is around 70°.29 Water droplets on the surface of a material whose contact angle is much less than 70° prevents protein adsorption, while denaturation of the protein causes it to lose its ability to adhere to cells on a material whose contact angle is much more than 70°. Although the hydrogel itself contains water inside and is expected to be a hydrophilic surface, some hydrogel contact angles were measured. For example, the water contact angle of a hydrogel composed of poly(allylamine) hydrochloride was in the range of 30° and 40°.30 To investigate the basic surface characteristics of PNVA gels, contact angles with ultrapure water were measured at a swollen state at 25 °C, as shown in Figure 2. Initially, a more hydrophobic nature was expected for the PNVA gel due to the secondary amide structure. However, the
Figure 3. Results of protein uptake were calculated with each surface area on PAAm gel (a), PNVA gel (b), NVA/NVA DN gel (c), and NVA/ AAm DN gel (d) and calculated with each bulk volume of PAAm gel (e), PNVA gel (f), NVA/NVA DN gel (g), and NVA/AAm DN gel (h).
mean value of the contact angle on the PNVA gel was 35.5 ( 0.4° (Figure 2b), whereas the PAAm gel contact angle was 38.7 ( 1.8° (Figure 2a). Rearrangements of hydrophilic groups to water could occur, however, the difference would probably be due to the larger amount of water occupying the surface area for the PNVA gel because the swelling ratio of the PAAm gel (9.1) was smaller than that of the PNVA gel. On the other hand, contact angles on both DN gels are apparently different from those on PNVA and PAAm gels with statistically significant differences (p < 0.01). Both DN gels (Figure 2c,d) tended to be more hydrophilic than PAAm gels but had similar swelling ratios. This hydrophilic character of DN gels is probably due to the twice glass contact effects during hydrogel preparation,
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Figure 4. Cell morphologies of fibroblast after 8 days on PAAm gel (a), PNVA gel (b), NVA/NVA DN gel (c), NVA/AAm DN gel (d), and after 4 days on plastic disk (e). White bar indicates 100 µm.
regardless of first network (PNVA gel and PAAm gel). When a second polymerization was achieved, hydrogel disks were tightly compressed by double glass plates (1.0 mm thickness). Glass contact sides of hydrogels possess more hydrophilic characteristics than those inside because of the orientation of hydrophilic groups of monomers to the glass phase during hydrogel preparations.30 BSA, BγG, and BPF are common proteins used to evaluate protein adsorption. As expected from the contact angles, only a small amount of protein adsorbed onto PNVA gels (Figure 3b) compared to those found on PAAm gels, although contact angles with ultrapure water and medium culture could be different (Figure 3a). Poor protein adsorption characteristics of PNVA gels are supported by Sakuma et al.18,19 The authors reported that NVA nanoparticles could not catch peptides, whereas poly(methacrylic acid), poly(acrylamide), and poly(N-isopropylacrylamide) were used as sCT adsorption materials in rat gastrointestinal tracts. However, both NVA/NVA DN gels and NVA/AAm DN gels contained more albumin and globulin than PNVA gel (p < 0.05) (Figure 3c,d). Assuming that protein exists only on the surface, PNVA gel and NVA/NVA DN gel, which were composed of the same chemical compound, would possess the similar value on protein adsorption (Figure 3b,c). However, NVA/NVA DN gel shows much protein adsorption in spite of more hydrophilic surface character. This result implies that the amount of protein detected by the BCA assay would come from the amount of protein adsorbed on the surface and also inside of the hydrogel. Adsorbed protein was estimated on area and volume, which were calculated with actual thickness and size after hydrogels were swollen (Figure 3). Generally, protein adsorption seems to be affected by the swelling ratio (Table 1). Assuming that the BSA concentration inside the hydrogel was the same as the prepared concentration (4.5 mg/mL), about 4500 µg/mL should be detected in each hydrogel (Figure 3e-h). However, the observed protein absorptions were less than 400 µg/mL. Furthermore, PNVA gel, which possesses the largest swelling ratio (the
highest volume), kept the smallest value. Therefore, not only volume but also polymer density inside hydrogels might be important in order to capture protein, and the different protein concentration would exist inside the hydrogel. In other words, DN gels seemed to uptake the protein readily, but there might appear to be a gradient of protein distribution inside of gels, although the detailed distribution remained unclear. Because PAAm gels have not been considered to be strong cell adhesion materials,22 PNVA gels were also predicted to be poor cell adhesion materials. Therefore, the PNVA gel could be used as a novel material in which less cell adhesion is required for biocompatibility such as the replacement of gauze and cushions for external injuries. However, to maximally apply its amphiphilic characteristics, cell adhesive PNVA gels would be more useful as drug delivery systems. Cell morphologies on hydrogels and on plastic disk (Cell Disk LF1, Sumitomo Bakelite Co., Ltd.) are shown in Figure 4. Although cell adhesion on hydrogels is less than that on plastics, most of the cells on hydrogels were round with partially spread morphologies. Interestingly, more bulk colonies appear on the NVA/NVA DN gel than on PNVA and NVA/AAm DN gels, implying no cellular degradation and more proliferation. (Figure 4). Figure 5 shows the number of adhesive cells after 8 days of cell culturing, which includes the results of cell death and cell proliferation. As expected from contact angle and protein uptake measurements results, the adhesion number of the PNVA gel remains modest (Figure 5b). This result is also supported from the same tendency that the PAAm gel has poor interactions with polypeptides.22 Interestingly, the number of adherent cells increased on the NVA/NVA DN gel as compared with the PNVA gel (Figure 5b,c) (p < 0.01). With the use of the DN gel, a more suitable protein sponge effect might be achieved near the surface. Although more cells were cultured on the NVA/ NVA DN gel than on the PNVA gel, the NVA/AAm DN gel was comparable to the PNVA gel (p > 0.1). While the total
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which are used as biomaterials, could also be important because supplemental changes compared to the original single network hydrogels depend on second monomer and DN structures. The generality of such DN effects on cell affinity are currently under investigation using various components. Acknowledgment. . This work was partially supported by KAKENHI: A Grand-in-Aid for Young Scientists (start-up) (19850014). Supporting Information Available. Photos of PNVA gels, NVA/NVA DN gels, and NVA/AAm DN gels. Cell morphologies on plastic after 2, 4, and 6 days. This material is available free of charge via the Internet at http://pubs.acs.org. Figure 5. Fibroblast cells adhesion after 8 days on PAAm gel (a), PNVA gel (b), NVA/NVA DN gel (c), and NVA/AAm DN gel (d) (n ) 3). Statistically significant differences (*p < 0.01) were obtained using a two-sample t test for each comparison. NS ) no significant difference.
number was small, significant difference was observed (n ) 3). This difference was probably due to the large uptake effect of protein on the NVA/NVA DN gel, and a homogeneously constructed secondary network with the same NVA monomer would be expected, although the NVA/AAm DN gel looked cloudy, which implied a partially crystallized microstructure (Supporting Information, Figure S1). In other words, results from cell adhesion experiments reflect both contact angle and protein uptake tendencies. Although the detailed reasons are still unknown, the bioaffinity of the DN gel could be controlled by changing the surface characteristics, the cross-linking density inside, and partial crystalline microstructures by the introduction of a secondary network. The larger swelling ratio of PNVA gels (14.4) than NVA/NVA DN gels (9.9) and NVA/AAm DN gels (9.0) might directly lead to a more hydrophilic nature and less protein uptake; however, we insist that the subsequent cell adhesion looks anomalous in spite of the similar swelling ratio between NVA/NVA DN and NVA/AAm DN gels. For DN gels, cell affinity depends not only on the swelling ratio but also on each component, i.e., surface and inside structures created in secondary networks. Therefore, DN is a potential approach for tuning PNVA gel features on biocompatibility. Further investigations on the DN approach using other hydrogels with swelling ratios and differences to improve cell affinies are currently under way.
Conclusions Although mouse fibroblast cells were cultured on hydrogel surfaces without harm, PNVA gels basically showed low cell adhesion properties with poor protein uptake. Introducing a DN structure, however, held more proteins in spite of small contact angles. The resultant biocompatibility of NVA/NVA DN gels improved, whereas NVA/AAm DN gels were comparable with PNVA gels. In other words, DN gels could provide diverse contact angles and protein uptake in hydrogels, which lead to controlled cell adhesion. Thus, an aprotic amphiphilic PNVA gel could be available as either a more or less cell adhesive material. DN approaches to modify hydrogel characteristics,
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BM701221C
