Research Article www.acsami.org
Janus Nanocomposite Hydrogels for Chirality-Dependent Cell Adhesion and Migration Andisheh Motealleh and Nermin Seda Kehr* Physikalisches Institut and CeNTech, Westfälische Wilhelms-Universität Münster, Heisenbergstraße 11, D-48149 Münster, Germany S Supporting Information *
ABSTRACT: Recently, there has been much interest in the chiralitydependent cell affinity to enantiomorphous nanomaterials (NMs), since, at the nanoscale level, enantiomers of (bio)molecules have different effects on cell behaviors. In this respect, this study used enantiomorphous NMs with which to generate the Janus nanocomposite (NC) hydrogels as multifunctional biomaterials for studying chirality-dependent cell adhesion and cell migration. These Janus NC hydrogels possess two enantiomorphous NC hydrogels, in which the different halves of the hydrogel contain the opposite enantiomers of a biopolymer functionalized nanomaterials. Thus, the enantiomers contact each other only at the midline of the hydrogel but are otherwise separated, yet they are present in the same system. This advanced system allows us to simultaneously study the impact that each enantiomer of a biopolymer has on cell behavior under the same reaction conditions, at the same time, and using only a single biomaterial. Our results show that cells have higher affinity for and migrate toward the part of the Janus NC hydrogel containing the biopolymer enantiomer that the cells prefer. KEYWORDS: nanocomposite hydrogels, chiral nanomaterials, cell adhesion, cell migration, periodic mesoporous organosilica
■
INTRODUCTION One of the basic goals of tissue engineering is to generate biomaterials that can mimic native extracellular matrix (ECM) characteristics and native tissue functions so that these materials can be used to repair or replace damaged organs or tissues.1,2 Nanocomposite (NC) hydrogels3−6 as artificial scaffolds have been used as alternatives to hydrogels in tissue engineering applications because of their enhanced chemical and physical properties compared to hydrogels.7−14 These improved properties have been achieved by reinforcing (soft) polymer matrix of hydrogels with (hard) nanomaterials (NMs). Therefore, NC hydrogels can exhibit the various advantageous characteristics associated with both NMs (e.g., size, shape, surface functionality, electrical, optical, and mechanical properties) and polymeric hydrogel networks (e.g., elasticity, hydrophilicity, porosity, high water absorption).8,12 Recently, studies have started investigating the chiralitydependent cell affinity to enantiomorphous nanomaterial surfaces15−20 (as alternative to surfaces that display chirality at the molecular level)21−23 because chirality at the nanoscale, can mimic at certain extent the physiochemical environment of ECM. To investigate this, we recently described the first example of enantiomorphous NC hydrogels prepared by silicabased nanomaterials functionalized with the enantiomers of bioactive molecules.18 The prepared multifunctional NC hydrogel scaffolds simultaneously provide nano(micro)topography and chirality in a 3D environment like in native tissue. We used the stereochemistry of nanomaterial surfaces to control the affinity of cells to the 3D NC hydrogel and demonstrated subsequent chirality-dependent cell enrichment. In another contribution, Zhang and Liu et al.19 prepared a chiral supramolecular gel from chiral nanofibers and found © 2017 American Chemical Society
chirality-dependent protein adsorption on the respective chiral nanostructures. They demonstrated that proteins interacted differently on enantiomorphous nanofibers, and these interaction differences on chiral nanostructures were significantly greater than the interaction differences seen on structures that display chirality at the molecular level. Besides these few examples of hydrogels prepared using chiral nanoscaled materials, little is known about the effects of chiral surfaces, especially at the nano(micro)meter-scale, on cellular behavior (e.g., adhesion, migration, differentiation) in 3D biomaterial networks, even though individual cells possess internal chirality24 and can sense external chirality in the 3D ECM, which has nano(micro)scale topography. In addition, these studies used two separate NC hydrogels, each use NMs functionalized with different enantiomer of biomolecules (NC “homo”-hydrogels). Therefore, studies have not yet been able to compare the effects of NMs having different functionality (enantiomer of biomolecules) on cell behavior at the same time and under the same reaction conditions using only single NC hydrogels (NC “hetereo-hydrogels”) prepared by the combination of two NC enantiomorphous hydrogels. In this context, we aimed to surpass the current state of the art for studying cell-chiral NM interactions in 3D NC hydrogels, which has been limited to using NC “homo”hydrogels. In this contribution, we prepared a type of NC hydrogel, Janus NC hydrogels (NC “hetereo”-hydrogels), as an advanced system for studying the effects chirality on cell behavior. Janus NC hydrogels were prepared by combining, Received: July 24, 2017 Accepted: September 7, 2017 Published: September 7, 2017 33674
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces DXP
PMO-OH-Alg for J-1, Alg and DXPPMO-PLL-Alg for J-2, Alg and PMO-PDL-Alg for J-3, DXPPMO-PLL-Alg and DXPPMO-OH-Alg for J-4, DXPPMO-PDL-Alg and DXPPMO-OH-Alg for J-5, DXPPMOPLL-Alg and DXPPMO-PDL-Alg for J-6 (0,2 mL) were placed side by side. After they were freeze-dried, J-1, J-2, J-3, J-4, J-5, and J-6 scaffolds were obtained. Cell Experiments in Alg, DXPPMO-OH-Alg, DXPPMO-PLL-Alg, DXP PMO-PDL-Alg, DXPZeo-OH-Alg, DXPZeo-PLL-Alg, and DXPZeoPDL-Alg Scaffolds in Serum-Containing and Serum-Free Media. The cells were carefully thawed and suspended in 10% fetal bovine serum (FBS)-containing cell culture media (RPMI 1640) for cell experiments in serum media or in RPMI 1640 (no FBS) for cell experiments in serum-free media. Then, the cells were seeded into the hydrogel scaffolds (approximately 10 000 cells for each scaffold). The scaffolds were covered with respective cell culture media (1 mL) and incubated for 1 and 4 days at 37 °C and 5% CO2 in an incubator. After the incubation periods, scaffolds were washed twice with phosphatebuffered saline (PBS) to remove nonadhered cells. Subsequently, scaffolds were transferred to another cell culture plate and treated with EDTA (0.04% w/v in PBS, without Ca2+/Mg2+) with gentle mixing. The cells were counted immediately by using a Neubauer chamber (trypan blue solution was used to detect dead cells). Protein Adsorption Experiments. The amount of serum proteins adsorbed on the Alg, DXPPMO-OH-Alg, DXPPMO-PLL-Alg, and DXPPMO-PDL-Alg; J-4, Janus-(DXPPMO-PLL-Alg|DXPPMO-OHAlg); J-5, Janus-(DXPPMO-PDL-Alg|DXPPMO-OH-Alg); and J-6, Janus-(DXPPMO-PLL-Alg|DXPPMO-PDL-Alg) was measured by the BCA (bicinchoninic acid) protein assay kit according to the manual instruction. The respective (Janus) NC hydrogels were incubated in the serum containing cell culture medium (10% FBS + RPMI 1640) (1 mL) for 1 d at 37 °C and 5% CO2. After incubation time, (Janus) NC hydrogels were placed in empty cell culture plates and washed with water × 3 times to remove loosely adsorbed proteins. Then Alg, DXP PMO-OH-Alg, DXPPMO-PLL-Alg, DXPPMO-PDL-Alg, and the two sides of Janus NC hydrogels were separately dissolved in the working reagent of the BCA protein assay kit and the amount of adsorbed proteins were quantified according to the manual instruction. Cell Experiments in J-1, J-2, J-3, J-4, J-5, and J-6 Scaffolds. The cells were carefully thawed and suspended in their specific medium (10% FBS + RPMI 1640). Then the cells were seeded homogeneously onto the hydrogel scaffolds (approximately 20 000 cells). The scaffolds were covered with cell culture media (1 mL) and incubated for 1 and 4 days at 37 °C and 5% CO2 in an incubator. After the incubation periods, scaffolds were washed twice with phosphate-buffered saline (PBS) to remove nonadhered cells. Subsequently, J-1, J-2, J-3, J-4, J-5, and J-6 scaffolds were separated into their parts (before cell experiments the small section of the middle part of the scaffolds were marked with colored plastic to distinguish the individual hydrogel parts of J1−J6). These parts were transferred to another cell culture plate and treated with EDTA (0.04% w/v in PBS, without Ca2+/Mg2+) with gentle mixing. The cells were counted immediately by using a Neubauer chamber (trypan blue solution was used to detect dead cells). Cell Migration Experiments in J-3 and J-6 Scaffolds. The cells were carefully thawed and suspended in their specific medium (10% FBS + RPMI 1640). Then the cells were seeded into only one part of the hydrogel scaffold (Alg of J-3, DXPPMO-PLL-Alg of J-6) (approximately 10 000 cells) (Figure S5). The scaffolds were covered with cell culture media (1 mL) and incubated for 10 min, 1, 3, and 6 days at 37 °C and 5% CO2 in an incubator. After the incubation periods scaffolds were washed twice with PBS to remove nonadhered cells. Subsequently, J-3 and J-6 scaffolds were separated into their parts (Alg and DXPPMO-PDL-Alg of J-3, DXPPMO-PLL-Alg, and DXPPMOPDL-Alg of J-6). These parts were transferred to another cell culture plate and treated with EDTA (0.04% w/v in PBS, without Ca2+/Mg2+) with gentle mixing. The cells were counted immediately by using a Neubauer chamber (trypan blue solution was used to detect dead cells). Reverse Cell Migration Experiments in J-6 Scaffold. The cells were carefully thawed and suspended in their specific medium (10% FBS +
side-by-side, two different NC hydrogels that use the same type of embedded NM, but where the NM particles were functionalized with different enantiomers of a biopolymer. These advanced NC hydrogels were used to simultaneously study and compare how the different enantiomers of biopolymers at NM surfaces affect cell behavior (e.g., adhesion, migration) in a 3D NC hydrogel environment under exactly the same reaction conditions and using a single biomaterial. We showed that cells differentiate the type of enantiomers on the NM surface and show different affinity for the resulting Janus NC hydrogel scaffolds. We used the cells’ preference for one enantiomer of a biopolymer to control the migration of cells toward the Janus NC hydrogel section that contained NMs functionalized with the biopolymer enantiomer that the cells preferred. As representative examples of NMs to use in the Janus NC hydrogels, we chose periodic mesoporous organosilica (PMO)25−28 and zeolite L.29 We previously successfully functionalized the external and internal surface of zeolite L30,31 and PMO25 and incorporated them into NC hydrogels for cell adhesion and surface-mediated drug delivery applications.18,32 In this work, the external surface of PMOs and zeolites were functionalized with the biodegradable chiral biopolymers poly-L-lysine (PLL) and poly-D-lysine (PDL), as both PLL and PDL are typical biopolymers used to coat surfaces for cell attachment and cellular uptake.31−34
■
DXP
EXPERIMENTAL SECTION
Materials. Poly-L(D)-lysine (PLL/PDL), hexadecyltrimethylammonium bromide (CTAB, 98%), 1,2-bis(trimethoxysilyl)ethane (BTME, 96%), N,N′-bis(2,6-dimethylphenyl)perylene-3,4,9,10-tetracarboxylicdiimide (DXP), paraformaldehyde (PFA), and trypsin were purchased from Sigma-Aldrich. Toluene, ethanol (absolute for analysis), ammonia solution (32%, pure), and hydrochloric acid (32%, for analysis), were purchased from Merck. Trypan blue solution was purchased by Life Technologies GmbH. Phalloidine Alexa Fluor488 was purchased by Invitrogen. 4′,6-Diamidino-2-phenylindoledihydrochloride (DAPI) was acquired from Polysciences Europe GmbH. BCA (bicinchoninic acid) protein assay kit was purchased by Thermo Scientific. The cell medium (RPMI 1640) supplemented with 10% (v/ v) fetal bovine serum (FBS) was obtained from Biochrom, Germany. Primary dermal fibroblast; normal, human, adult (ATCC PCS-201012) cells were purchased from ATCC. General Procedure for the Preparation of Alg, DXPPMO-OHAlg, DXP PMO-PLL-Alg, DXP PMO-PDL-Alg, DXP Zeo-OH-Alg, DXP Zeo-PLL-Alg, and DXPZeo-PDL-Alg Hydrogels. Alg Hydrogel. Stock solutions of alginate (1.25 g) in 100 mL of double-distilled water and calcium D-gluconate monohydrate (1.00 g) in 100 mL of doubledistilled water were prepared. 0.7 mL of alginate stock solution was calcium cross-linked by adding a 0.3 mL stock solution of calcium Dgluconate monohydrate, while homogenizing the solution to obtain homogeneous calcium ion distribution and cross-linking. DXP PMO-OH-Alg, DXPPMO-PLL-Alg, DXPPMO-PDL-Alg, DXPZeo-OHAlg, DXPZeo-PLL-Alg, and DXPZeo-PDL-Alg hydrogels. The suspensions of PMOs/zeolites (0.3 mg) in calcium D-gluconate monohydrate (0.3 mL) were sonicated for 20 min and then added into the alginate solution (0.7 mL) during vortexing. This resulted in 0.03 w/v % PMO/zeolite in alginate hydrogel. General Procedure for the Preparation of Alg, DXPPMO-OHAlg, DXP PMO-PLL-Alg, DXP PMO-PDL-Alg, DXP Zeo-OH-Alg, DXP Zeo-PLL-Alg, and DXPZeo-PDL-Alg Scaffolds. The cross-linked Alg and NC hydrogels (0.2 mL) were transferred into a Teflon container, frozen at −80 °C for 16 h, and then lyophilized with freezedryer for 16 h. General Procedure for the Preparation of J-1 to J-6 Scaffolds. Alg, DXPPMO-OH-Alg, DXPPMO-PLL-Alg, and DXPPMOPDL-Alg hydrogels were prepared. Then the hydrogel couples Alg and 33675
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces RPMI 1640). Then the cells were seeded into the DXPPMO-PDL-Alg part of J-6 (approximately 10 000 cells) (Figure S6). Then J-6 was covered with cell culture media (1 mL) and incubated for 10 min, 1 day, 3 days and 6 days at 37 °C and 5% CO2 in an incubator. After the incubation periods J-6 was washed twice with PBS to remove nonadhered cells. Subsequently, J-6 was separated into its parts (DXPPMO-PLL-Alg and DXPPMO-PDL-Alg). These parts were transferred to another cell culture plate and treated with EDTA (0.04% w/v in PBS, without Ca2+/Mg2+) with gentle mixing. The cells were counted immediately by using a Neubauer chamber (trypan blue solution was used to detect dead cells). General Procedure for the Costaining of Cells. Cells in Alg, DXP PMO-OH-Alg, DXPPMO-PLL-Alg, and DXPPMO-PDL-Alg were fixed with 4% PFA solution after 1 d incubation time. After treatment of cells with PFA for 10 min cells were washed twice with PBS and kept in 0.1% Triton X-100 in PBS for 10 min and afterward in 3% bovine serum albumin (BSA), in PBS for 20 min. Then cells were stained with Phalloidin Alexa Fluor 488 (for f-actin staining) for 40 min, in the dark at room temperature, and washed twice with PBS. Finally, cell nucleus was stained with DAPI and washed twice with PBS.
■
scaffolds (Figure S3). The particle distribution, determined by ImageJ’s multipoint tool, was similar in each NC hydrogel scaffolds (Table S4). These results indicated that the respective NC hydrogel scaffolds were comparable. As shown in Tables S5−S7 the scaffolds with presence of PMO and also functionalized PMO exhibit a higher swelling capacity, high equilibrium water content, and less degradation in comparison to alginate scaffold. The incorporation of hydrophilic porous PMOs into hydrogel network improves the hydrophilicity and mechanical stability of the hydrogel network and increases the water diffusion and decreases the degradation of the respective NC hydrogel scaffolds. The weight loss (%) of NC hydrogel scaffolds was less than 1% after 4 days (Table S7) confirming the stability of our system at 37 °C in the presence of cells for 1 day and 4 days incubation time. Pore size and porosity measurements showed (Table S8) that the samples with presence of PMO have higher porosity and lower pore size in comparison with alginate scaffold itself. Higher porosity might be because of enhancing the stability of the structure by presence of PMO within cross-linked network of hydrogel. Cell Experiments in Alginate and NC Hydrogel Scaffolds. Cell adhesion experiments were carried out in alginate and DXPPMO-OH/PLL/PDL-Alg and DXPZeo-OH/ PLL/PDL-Alg scaffolds, and primary fibroblast cells were selected for the experiments. Fibroblasts are mechanosensitive cells.36 Mechanosensitivity plays an important role in migration and adhesion of fibroblast cells and can be enhanced with certain levels of soluble calcium ions.37,38 Therefore, we did all cell experiment using cell culture media that contains calcium ion. To investigate only the impact of chirality on cell behavior we did not add additional calcium source to the cell culture media. Our cells experiments (Figure 1, Table 1) showed that cells had higher affinity for DXPPMO-PLL/PDL and DXPZeoPLL/PDL than they did for Alg and DXPPMO/Zeo-OH-Alg at 1 day and 4 days of incubation. This indicates that NMs and biopolymer-coated NMs positively influence cell affinity for the 3D NC hydrogel scaffolds. When we compare the impact of chirality on cell adhesion, we observed that cells had greater affinity for DXPPMO/DXPZeo-PDL-Alg than DXPPMO/DXPZeoPLL-Alg, showing that cell adhesion in NC hydrogels prepared by using enantiomerically functionalized PMOs and zeolites is chirality dependent. After an extension of the incubation time from 1 to 4 days, we observed similar cell behavior to each NC hydrogel scaffold, but in most cases, we found fewer cells after 4 days of incubation than after 1 day (Figure 1, Table 1) most probably due to the decrease in cell viability. Moreover, the difference between D X P PMO/ D X P Zeo-PDL-Alg and DXP PMO/DXPZeo-PLL-Alg was less significant after 4 days than after 1 day of incubation. In addition, we compared the effect of the two different NMs, which have different shapes and sizes, on cell behavior within the NC hydrogel scaffolds. We observed that cells showed similar affinity to noncoated and biopolymer-coated PMOs and zeolites that were inserted into the hydrogel scaffolds, showing that the different NM sizes and shapes did not significantly affect cell behavior within the 3D hydrogel network. Furthermore, in almost all hydrogel scaffolds, we observed a slight decrease in cell viability with increasing incubation time (Table 1). In general, cell viability was higher in PMO-embedded NC hydrogel scaffolds than in the zeolite-embedded ones, indicating that PMOs are less cytotoxic than zeolite within the NC hydrogels. Upon interacting with serum-containing media, NM surfaces become coated with proteins. The formed NM-protein
RESULTS AND DISCUSSION
Preparation of NC Hydrogel Scaffolds. Sphere-shaped PMOs18 and disc-shaped zeolite L particles29 were synthesized, and the pores of PMOs11 and the channels of zeolites35 were loaded with N,N′-bis(2,6-dimethylphenyl)perylene-3,4,9,10tetracarboxylic diimide (DXP), a nonwater-soluble fluorescent dye molecule, to get DXPPMO-OH and DXPZeo-OH according to the literature method. Subsequently, negatively charged surfaces of DXPPMO-OH and DXPZeo-OH were coated with the cationic biopolymer enantiomers PLL and PDL to obtain fluorescence dye molecule-loaded chiral NMs (DXPPMO-PLL/ PDL and DXPZeo-PLL/PDL) using a literature procedure.31 The amount of PLL and PDL was found almost in same amount on DXPPMO/Zeo-PLL and DXPPMO/Zeo-PDL (Table S1). DXP PMO-PLL/PDL and DXPZeo-PLL/PDL were characterized also by dynamic light scattering (DLS), scanning electron microscopy (SEM), infrared spectroscopy (IR) and zetapotential measurements (see Figures S1 and 2 and Table S2). The prepared DXPPMO/Zeo-OH, DXPPMO/Zeo-PLL, and DXP PMO/Zeo-PDL were embedded into alginate hydrogels and then freeze-dried to obtain the respective NC hydrogel scaffolds (DXPPMO-OH/PLL/PDL-Alg and DXPZeo-OH/PLL/ PDL-Alg) (see Supporting Information). The respective alginate scaffold and NC hydrogel scaffolds were characterized using SEM. The SEM images show the 3D porous Alg network and the zoomed-in images show the PMOs and zeolites embedded into the Alg scaffold wall (Figure S3). DXPPMO-OHAlg, DXPPMO-PLL-Alg, and DXPPMO-PDL-Alg were further characterized. These NC hydrogel scaffolds were utilized in most of the cell experiments (see Cell Experiments in Alginate and NC Hydrogel Scaffolds section). The quantitative amount of PMOs in each NC hydrogel scaffolds, particle distribution, swelling ratio, equilibrium water content, degradation behavior, porosity and pore size of the NC hydrogel scaffolds were analyzed (see Tables S3−S8). The quantitative amount of e.g. DXP PMO-OH, DXPPMO-PLL, and DXPPMO-PDL in each hydrogel was measured with fluorescence spectrometry to confirm that each NC hydrogels has almost same amount of particles (Table S3). In addition, we determined the DXPPMOOH, DXPPMO-PLL, and DXPPMO-PDL distribution on 1 μm2 area of Alg using SEM image of the respective NC hydrogel 33676
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces
NC hydrogels. Additionally, we observed no significant difference in cell adhesion between DXPPMO/DXPZeo-PDLAlg and DXPPMO/DXPZeo-PLL-Alg. These results show that protein adsorption onto enantiomerically functionalized NMs does significantly affect cell-chiral surface interactions. To strengthen these findings, we determined the amount of adsorbed serum proteins on Alg, DXPPMO-OH-Alg, DXPPMOPLL-Alg, and DXPPMO-PDL-Alg using the BCA (bicinchoninic acid) protein assay kit (Table S10). PMOs were used as NMs for further experiments because the difference in cell density between DXPPMO-PLL-Alg and DXPPMO-PDL-Alg was more distinct than that in between DXPZeo-PLL-Alg and DXPZeoPDL-Alg. Our results showed that the amount of adsorbed proteins on DXPPMO-PDL-Alg is approximately 10% higher than that on DXPPMO-PLL-Alg. Furthermore, serum proteins exhibited a stronger adsorption on the NC hydrogels than that on Alg showing that the chirality and nanostructure of PMO influence the protein adsorption on the respective surfaces. The reason for the differentiated adsorption behavior of proteins on chiral surfaces can be attributed to the specific stereoselective interactions between proteins and chiral surfaces. Therefore, proteins adsorb differently on surfaces functionalized with Land D-molecules, they create new stereochemistries on the respective chiral surfaces, which in turn influence cell affinity for those surfaces. Overall, these results indicate the indirect effect of surface functionality at PMO surfaces on cell behavior. Recently, the similar indirect effect of functional groups on cell behavior such as stem cell differentiation by tuning protein adsorption and thus cell spreading was described by Ding et al.39 It is important to note that PLL is more easily degraded by cells than PDL as described by Tung et al.40,41 Therefore, the enantiomer-specific degradation of biopolymers on PMOs by cells can be considered as one of the reason for the observed difference in the amount of cells in DXPPMO-PLL-Alg and DXP PMO-PDL-Alg. However, if the degradation is the most likely reason then it is expected to observe the similar difference in the amount of cells between DXPPMO-PLL-Alg and DXP PMO-PDL-Alg in serum-free media (Table S9) like in serum-containing media (Table 1) conditions. Therefore, the difference between the cell experiments in serum-containing and serum-free media indicates that the differential cell behavior between DXPPMO-PLL-Alg and DXPPMO-PDL-Alg is most likely mediated by the stereospecific interaction between chiral PMOs and proteins and the amount of proteins adsorbed on the chiral surfaces rather that the chirality dependent degradation of biopolymers. Preparation of Janus NC Hydrogel Scaffolds and the Respective Cell Experiments. After analyzing cell affinity for the NC homo-hydrogels that contained one type of enantiomerically functionalized NM, we prepared Janus NC Hydrogels by using two different types of enantiomerically functionalized PMOs.
Figure 1. Number of cells (×103) in Alg, DXPDXPPMO-PLL-Alg, DXP PMO-PDL-Alg, DXPZeo-OH-Alg, DXPZeo-PLL-Alg, and DXPZeoPDL-Alg in serum-containing media after incubation times of (A) 1 and (B) 4 days [The number of repeated experiments (N = 3; *, data show significant differences; t test, p ≤ 0,05)].
complex depends on the surface chemistry of NMs, and this complex affects the bioactivity of the NMs and their interaction with cells. Given that we15,18,21 and other groups20,22,23 have demonstrated that chirality-dependent cell adhesion is influenced by the protein adsorption on chiral NM surfaces, we also performed our experiments in serum-free media. This enabled us to study the how protein adsorption onto the PDLand PLL-coated NMs embedded in the NC hydrogel scaffolds affected cells’ ability to differentiate between the enantiomers (Table S9). In serum-free media, we found that fewer cells adhered to the NC hydrogels and they had lower cell viability (Table S9) than in serum-containing media (Table 1). Yet, cells still showed high affinity for the biopolymer-coated NMs embedded in the
Table 1. Quantitative Numbers (×103) of Fibroblast Cells and Cell Viabilitya after 1 and 4 Days Incubation Time (37 °C) in the Alg, DXPPMO-OH-Alg, DXPPMO-PLL-Alg, DXPPMO-PDL-Alg, DXPZeo-OH-Alg, DXPZeo-PLL-Alg, and DXPZeo-PDL-Alg in Serum Mediab Alg 1 day 4 days a
2.5 ± 0.4 (96%) 2.3 ± 0.6 (79%)
DXP
PMO-OH-Alg
4.3 ± 0.6 (90%) 5.2 ± 0.7 (85 4%)
DXP
PMO-PLL-Alg
7.9 ± 0.2 (94%) 5.9 ± 0.4 (81 4%)
DXP
PMO-PDL-Alg
11.5 ± 0.8 (96%) 7.1 ± 0.7 (87 4%)
DXP
DXP
5.7 ± 0.6 (71%) 6.0 ± 0.1 (44%)
8.0 ± 1.0 (79%) 7.3 ± 0.6 (64%)
Zeo-OH-Alg
Zeo-PLL-Alg
DXP
Zeo-PDL-Alg
10.1 ± 0.6 (78%) 8.7 ± 0.6 (73%)
In parentheses. bN = 3; *, data show significant differences; t-test, p ≤ 0.05. 33677
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces We first separately prepared Alg, DXP PMO-OH-Alg, PMO-PLL-Alg, and DXPPMO-PDL-Alg hydrogels (Figure 2A). Then, various different hydrogel couples, for example, Alg DXP
Figure 3. (A) Cell suspension in culture media was homogeneously added onto the Janus NC hydrogel. (B) The whole system was incubated for 1 day and 4 days. (C) After each incubation time, the two parts of the Janus NC hydrogel were separated and cells in each part were counted.
Figure 2. (A) DXPPMO-embedded Alg (red) and Alg (colorless) hydrogels. (B) Janus NC hydrogel prepared by connecting Alg and DXP PMO-embedded Alg. (C) Janus NC hydrogel scaffold prepared by connecting Alg and DXPPMO-embedded Alg.
and DXPPMO-PDL-Alg, DXPPMO-PLL-Alg and DXPPMO-PDLAlg, etc., were placed side by side to generate various Janus NC Hydrogels (Figure 2B). After they were freeze-dried (Figure 2C), we obtained these Janus NC hydrogel scaffolds: J-1, Janus(Alg | DXPPMO-OH-Alg); J-2, Janus-(Alg | DXPPMO-PLL-Alg); J-3, Janus-(Alg | DXPPMO-PDL-Alg); J-4, Janus-(DXPPMO-PLLAlg | DXPPMO-OH-Alg); J-5, Janus-(DXPPMO-PDL-Alg | DXP PMO-OH-Alg); J-6, Janus-(DXPPMO-PLL-Alg | DXPPMOPDL-Alg). Janus NC hydrogels were composed two NC hydrogels that used the same NM as a base (PMO) but that were functionalized with different functional groups, that is, the enantiomers of a biopolymer. In this study, the two sides of the Janus NC hydrogels were distinct from each other by either the presence or absence of NMs/functionalized NMs or by the type of functionalized biopolymer on the NMs (the L- or Dbiopolymer). Therefore, we used this advanced multifunctional system to simultaneously study and compare how the NMs and the different functional groups (biopolymer enantiomers) on the NM surfaces affect cell affinity for 3D NC hydrogels under exactly the same reaction conditions and on a single biomaterial. Such system is similar to the advanced micropatterned surfaces described by Ding et al. They described an elegant strategy42,43 based on microtransfer technique to prepare a variety of cell-adhesive microislands of various sizes on the same adhesion-resistant substrate surface to study systematically cell-material interactions.43,44 Cell experiments were performed in the respective Janus NC hydrogels for incubation times of 1 and 4 days at 37 °C. Cells were first suspended in their specific cell culture serum media and seeded homogeneously on the Janus NC hydrogels (Figure 3). After each incubation time, Janus NC hydrogels were separated into their two individual NC hydrogel parts and cells were counted in each part (Figure 3C). On the individual parts of the Janus NC hydrogels, we found similar trends in cell viability and cell affinity as were seen for the corresponding NC homo-hydrogels at 1 and 4 days of incubation (Figure 4 and Table 2). We observed higher cell affinity for DXPPMO-OH-Alg (in J-1) and DXPPMO-PLL/PDLAlg (in J-2 and J-3) than for Alg itself, and higher cell affinity for DXPPMO-PLL/PDL-Alg (in J-4 and J-5) than for DXPPMOOH-Alg. Additionally, we found a higher number of cells in DXP PMO-PDL-Alg than DXPPMO-PLL-Alg (in J-6). However, the total number of cells in each individual part of the Janus NC hydrogels (Table 2) was less than in the corresponding NC homo-hydrogels (Table 1). This most likely occurred because the cells were not directly seeded into the Janus NC hydrogels
Figure 4. Number of cells (×103) in individual parts of J-1, J-2, J-3, J4, J-5, and J-6 in serum-containing media after an incubation time of (A) 1 and (B) 4 days (N = 3; *, data show significant differences; t test, p ≤ 0,05).
(as they were in NC homo-hydrogels); rather, cells were homogeneously seeded into the media. Therefore, some of the cells in the media may have bypassed the Janus NC hydrogels and instead seeded onto the cell culture plate. Subsequently, the protein adsorption experiments were done with Janus NC hydrogel scaffolds. The results also showed that the different amount of adsorbed proteins on the two different parts of the Janus NC hydrogel scaffolds (Table S11). These results are consistent with the protein adsorption experiments on single NC hydrogels (Table S10). In cell experiments with the NC homo-hydrogels, cells were directly seeded into the NC hydrogel scaffolds, such that cells had only one possible hydrogel scaffold on which to grow. Conversely, in the Janus NC hydrogel experiments, cells encounter two alternative hydrogel scaffolds with different properties. Therefore, the Janus NC hydrogel system allows us to simultaneously observe how these different properties affect cell behavior under the same reaction conditions, at the same time, and while using only a single biomaterial [e.g., Janus(DXPPMO-PLL-Alg | DXPPMO-PDL-Alg)] instead of two separate biomaterials (e.g., DXPPMO-PLL-Alg and DXPPMOPDL-Alg). 33678
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces Table 2. Quantitative Numbers (×103) of Fibroblast Cells and Cell Viabilitya after 1 and 4 Days Incubation Time (37 °C) in the Both Sides of the J-1, J-2, J-3, J-4, J-5, and J-6 (N = 3)
a
the chirality-dependent cell behavior to direct cell migration toward the Janus NC hydrogel section containing PMOs functionalized with the biopolymer enantiomer that cells prefer. Cell Migration in Janus NC Hydrogel Scaffolds. Janus(Alg | DXPPMO-PDL-Alg) (J-3) and Janus-(DXPPMO-PLL-Alg | DXP PMO-PDL-Alg) (J-6) were used to study chirality-dependent cell migration. These constructs were prepared by combining Alg and DXPPMO-PDL-Alg (J-3) and DXPPMOPLL-Alg and DXPPMO-PDL-Alg (J-6). Cell were seeded into the Alg part of J-3 and into the DXPPMO-PLL-Alg part of J-6 and incubated for 10 min, 1, 4, and 6 days at 37 °C (Figure 5A, 5B). Thereafter, J-3 and J-6 were separated into their individual parts and cells in each part were counted (Figures 6A and 6B and Table 3A and 3B). After 10 min incubation, most of the cells were found in Alg part of J-3 and in the DXPPMO-PLL-Alg part of J-6 than in the DXP PMO-PDL-Alg parts (present in both J-3 and J-6). At longer incubation times, we observed a continuous decrease in the number of cells in the Alg and DXPPMO-PLL-Alg parts, while the number of cells continuously increased in DXPPMOPDL-Alg parts of J-3 and J-6. The difference in the number of cells on each side of the J-3 and J-6 constructs was more remarkable in J-3 than J-6; this probably occurred because the cells have less affinity for Alg than they do for DXPPMO-PLLAlg. Overall, we found that in J-3 and J-6, cells migrated toward the NC hydrogel constructs containing the functionalized biopolymers, and also toward those having PMO functionalized with the cell-preferred enantiomer of the biopolymer (DXPPMO-PDL-Alg); in other words, cells displayed biopolymer- and chirality-dependent cell migration.
DXP
J-1
Alg
1 day 4 days J-2
2.5 ± 0.5 (92%) 1.7 ± 0.3 (84%) Alg
3.2 ± 0.6 (88%) 3.4 ± 1.0 (83%) DXP PMO−PLL-Alg
PMO-OH-Alg
1 day 4 days J-3
2.9 ± 0.2 (97%) 1.6 ± 0.4 (83%) Alg
5.9 ± 0.8 (97%) 4.4 ± 0.9 (85%) DXP PMO-PDL-Alg
1 day 4 days J-4
2.4 ± 0.1 (92%) 1.7 ± 0.3 (83%) DXP PMO-PLL-Alg
7.8 ± 0.9 (98%) 5.4 ± 0.5 (84%) DXP PMO-OH-Alg
1 day 4 days J-5
5.3 ± 0.7 (92%) 4.2 ± 0.3 (81%) DXP PMO-PDL-Alg
3.4 ± 0.6 (94%) 3.1 ± 0.8 (80%) DXP PMO-OH-Alg
1 day 4 days J-6
7.9 ± 0.5 (97%) 5.6 ± 0.8 (87%) DXP PMO-PLL-Alg
3.2 ± 0.4 (94%) 3.1 ± 0.8 (89%) DXP PMO-PDL-Alg
1 day 4 days
5.2 ± 0.4 (97%) 4.0 ± 0.5 (85%)
7.9 ± 0.9 (92%) 5.3 ± 0.2 (89%)
In parentheses.
Overall, the cell-Janus NC hydrogel experiments again showed that cells have higher affinity for biopolymer-functionalized PMOs that are incorporated into hydrogels, and they are able to differentiate between which enantiomer is on the PMO surfaces. For the Janus NC hydrogels that contained e.g. both enantiomers located on opposite sides of the construct, we used
Figure 5. Cell suspension in culture media was added to (A) the Alg part of J-3, (B) the DXPPMO-PLL-Alg part of J-6, and (C) the DXPPMO-PDLAlg part of J-6. The number of cells in the both sides of J-3 and J-6 were counted after 10 min (i), 1 day (ii), 4 days (iii), and 6 days (iv). 33679
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces
seeded cells into the DXPPMO-PDL-Alg sides. We incubated the constructs for 10 min, 1 day, 4 days, and 6 days at 37 °C (Figure 5C). Thereafter, J-6 constructs were separated into their individual parts (DXPPMO-PLL-Alg and DXPPMO-PDLAlg), and cells in each part were counted (Figure 6C, Table 3C). The initial distribution of the total number of cells in DXP PMO-PLL-Alg and DXPPMO-PDL-Alg was 22% and 78%, respectively. But with longer incubation time, we observed a slight increase and then a decrease in the number of cells in DXP PMO-PLL-Alg. Conversely, in the DXPPMO-PDL-Alg part, the number of cells continiously increased over time, confirming our findings that cell affinity and migration depend on chirality. Additionally, cell viability in these experiments (Table 3) was consistent with the cell viability in NC homo-hydrogels (Table 1). The morphology of cells in the respective hydrogel networks were also determined by costaining of cell nucleus and actin filament. The fluorescence microscopy images show that cells form clusters and they have spherical shaped morphology in hydrogel network, as is commonly seen in 3D networks (Figure S4).18,21 The number of cells in the clusters is higher in DXP PMO-PDL-Alg than Alg and other NC hydrogels.
■
CONCLUSION In this study, we synthesized enantiomorphous NMs and used them to prepare NC homo-hydrogels and Janus NC hydrogels. The NC homo-hydrogels were prepared using one type of functionalized NM (either PMO or zeolite) embedded in an alginate hydrogel, while the Janus NC hydrogels were prepared by combining differently formulated NC hydrogels; namely, those that did or did not contain functionalized NMs (PMOs) or those that contained NMs (PMOs) functionalized with different biopolymers. Cell experiments in the NC homohydrogels showed that cells’ affinity to the respective hydrogel scaffolds was affected by the enantiomer of the biopolymer functionalized to the PMO/zeolite. This chirality-dependent cell affinity was more pronounced in serum-containing media. We determined that more serum proteins were adsorbed on DXP PMO-PDL-Alg than on DXPPMO-PLL-Alg. This chiralitydependent protein adsorption most probably led to a more cell adhesion on DXPPMO-PDL-Alg than on DXPPMO-PLL-Alg. These results show that protein adsorption is important for chirality to have an effect on NM surfaces, as this established stereochemistry affects cell affinity for the 3D NC hydrogels.
Figure 6. (A) Number of cells (×103) after 10 min, 1 day, 4 days, and 6 days incubation time (37 °C) in both sides of (A) J-3, (B) J-6 (cells were seeded in Alg and DXPPMO-PLL-Alg part of J-3 and J-6, respectively), and (C) J-6 (cells were seeded in the DXPPMO-PDL-Alg part of J-6) (N = 3; *, data show significant differences; t test, p ≤ 0,05).
To verify and thus strengthen our findings about chiralitydependent cell migration, we did another experiment, a reverse cell migration experiment. We prepared J-6 constructs and only
Table 3. Quantitative Numbers (×103) of Fibroblast Cells and Cell Viabilitya after 10 min, 1 day, 4 days, and 6 days Incubation Time (37 °C) in Both Sides of (A) J-3, (B) J-6 (Cells Were Seeded in Alg and DXPPMO-PLL-Alg Part of J-3 and J-6, Respectively), and (C) J-6 (Cells Were Seeded in the DXPPMO-PDL-Alg part of J-6) (N = 3) (A) J-3
10 min
1 day
4 days
6 days
Alg DXP PMO-PDL-Alg (B) J-6
8.8 ± 0.6 (100%) 0.6 ± 0.1 (100%) 10 min
5.0 ± 0.5 (90%) 3.2 ± 0.2 (80%) 1day
3.5 ± 0.7 (83%) 3.7 ± 1.1 (89%) 4 days
2.6 ± 0.8 (78%) 3.9 ± 0.5 (89%) 6 days
DXP
PMO-PLL-Alg PMO-PDL-Alg (C) J-6
8.8 ± 0.7 (100%) 0.7 ± 0.1 (100%) 10 min
8.3 ± 0.3 (97%) 2.0 ± 0.6 (93%) 1day
6.1 ± 1.2 (90%) 3.0 ± 0.5 (88%) 4 days
5.4 ± 0.9 (88%) 3.4 ± 1.2 (95%) 6 days
PMO-PLL-Alg PMO-PDL-Alg
2.2 ± 0.2 (100%) 7.6 ± 0.2 (100%)
3.0 ± 0.9 (99%) 8.1 ± 0.3 (91%)
3.3 ± 0.6 (90%) 8.2 ± 0.8 (96%)
1.7 ± 0.3 (83%) 9.0 ± 0.9 (93%)
DXP
DXP DXP
a
In parentheses. 33680
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces
(7) Shin, S. R.; Aghaei-Ghareh-Bolagh, B.; Dang, T. T.; Topkaya, S. N.; Gao, X.; Yang, S. Y.; Jung, S. M.; Oh, J. H.; Dokmeci, M. R.; Tang, X.; Khademhosseini, A. Cell-laden Microengineered and Mechanically Tunable Hybrid Hydrogels of Gelatin and Graphene Oxide. Adv. Mater. 2013, 25, 6385−6391. (8) Motealleh, A.; Kehr, N. S. Nanocomposite Hydrogels and Their Applications in Tissue Engineering. Adv. Healthcare Mater. 2017, 6, 1600938. (9) Wang, Q.; Mynar, J. L.; Yoshida, M.; Lee, E.; Lee, M.; Okuro, K.; Kinbara, K.; Aida, T. High-water-content Mouldable Hydrogels by Mixing Clay and a Dendritic Molecular Binder. Nature 2010, 463, 339−343. (10) Huang, Y.; Yao, M.; Zheng, X.; Liang, X.; Su, X.; Zhang, Y.; Lu, A.; Zhang, L. Effects of Chitin Whiskers on Physical Properties and Osteoblast Culture of Alginate Based Nanocomposite Hydrogels. Biomacromolecules 2015, 16, 3499−3507. (11) Kehr, N. S.; Prasetyanto, E. A.; Benson, K.; Ergün, B.; Galstyan, A.; Galla, H. J. Periodic Mesoporous Organosilica-Based Nanocomposite Hydrogels as Three-Dimensional Scaffolds. Angew. Chem., Int. Ed. 2013, 52, 1156−1160. (12) Gaharwar, A. K.; Peppas, N. A.; Khademhosseini, A. Nanocomposite Hydrogels for Biomedical Applications. Biotechnol. Bioeng. 2014, 111, 441−453. (13) Seda Kehr, N.; Riehemann, K. Controlled Cell Growth and Cell Migration in Periodic Mesoporous Organosilica/Alginate Nanocomposite Hydrogels. Adv. Healthcare Mater. 2016, 5, 193−197. (14) Thoniyot, P.; Tan, M. J.; Karim, A. A.; Young, D. J.; Loh, X. J. Nanoparticle−Hydrogel Composites: Concept, Design, and Applications of These Promising, Multi-Functional Materials. Adv. Sci. 2015, 2, 1400010. (15) Kehr, N. S.; Galla, H.-J.; Riehemann, K.; Fuchs, H. Selfassembled Monolayers of Enantiomerically Functionalized Periodic Mesoporous Organosilicas and the Effect of Surface Chirality on Cell Adhesion Behavior. RSC Adv. 2015, 5, 5704−5710. (16) El-Gindi, J.; Benson, K.; De Cola, L.; Galla, H.-J.; Seda Kehr, N. Cell Adhesion Behavior on Enantiomerically Functionalized Zeolite L Monolayers. Angew. Chem., Int. Ed. 2012, 51, 3716−3720. (17) Deng, J.; Wu, S.; Yao, M.; Gao, C. Surface-anchored Poly(acryloyl-L(D)-valine) with Enhanced Chirality-selective Effect on Cellular Uptake of Gold Nanoparticles. Sci. Rep. 2016, 6, 31595. (18) Kehr, N. S. Enantiomorphous Periodic Mesoporous Organosilica-Based Nanocomposite Hydrogel Scaffolds for Cell Adhesion and Cell Enrichment. Biomacromolecules 2016, 17, 1117−1122. (19) Lv, K.; Zhang, L.; Lu, W.; Liu, M. Control of Supramolecular Chirality of Nanofibers and Its Effect on Protein Adhesion. ACS Appl. Mater. Interfaces 2014, 6, 18878−18884. (20) Liu, G. F.; Zhang, D.; Feng, C. L. Control of Three-dimensional Cell Adhesion by the Chirality of Nanofibers in Hydrogels. Angew. Chem., Int. Ed. 2014, 53, 7789−7783. (21) Benson, K.; Galla, H.-J.; Kehr, N. S. Cell Adhesion Behavior in 3D Hydrogel Scaffolds Functionalized with D- or L-Aminoacids. Macromol. Biosci. 2014, 14, 793−798. (22) Qing, G.; Zhao, G.; Xiong, Y.; Lv, Z.; Jiang, F.; Liu, Y.; Chen, H.; Zhang, M.; Sun, T. Chiral Effect at Protein/Graphene Interface: A Bioinspired Perspective To Understand Amyloid Formation. J. Am. Chem. Soc. 2014, 136, 10736−10742. (23) Yao, X.; Hu, Y.; Cao, B.; Peng, R.; Ding, J. Effects of Surface Molecular Chirality on Adhesion and Differentiation of Stem Cells. Biomaterials 2013, 34, 9001−9009. (24) Yamanaka, H.; Kondo, S. Rotating Pigment Cells Exhibit an Intrinsic Chirality. Genes Cells 2015, 20, 29−35. (25) Benson, K.; Prasetyanto, Y. E. A.; Galla, H.-J.; Kehr, N. S. Selfassembled Monolayers of Bifunctional Periodic Mesoporous Organosilicas for Cell Adhesion and Cellular Patterning. Soft Matter 2012, 8, 10845−10852. (26) Inagaki, S.; Guan, S.; Fukushima, Y.; Ohsuna, T.; Terasaki, O. Novel Mesoporous Materials with a Uniform Distribution of Organic Groups and Inorganic Oxide in Their Frameworks. J. Am. Chem. Soc. 1999, 121, 9611−9614.
Similar cell experimental results were also obtained in the Janus NC hydrogels in which, for example, the two enantiomeric NC hydrogels (DXPPMO-PLL-Alg and DXPPMOPDL-Alg) were connected to each other. In this construct, the opposite enantiomers of the biopolymer were separated from each other, but were present together in the same system. Therefore, we were able to simultaneously observe the impact that each enantiomer had on cell behavior under the same reaction conditions, at the same time, and using only a single biomaterial [e.g., Janus-(DXPPMO-PLL-Alg | DXPPMO-PDLAlg)] instead of two (e.g., DXPPMO-PLL-Alg and DXPPMOPDL-Alg). Finally, we used this chirality-dependent cell behavior to direct cell migration toward the part of the Janus NC hydrogel containing the PMOs functionalized with the biopolymer enantiomer that the cells preferred. We envisage that such advances systems (the Janus NC hydrogels) should find applications in biomedical field (e.g., as biomedical device for cell enrichment and cell−cell separation or in tissue engineering).
■
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b10871. Experimental procedures of the synthesis, functionalization of PMOs and zeolites L; analytic characterization data (SEM, IR) and physicochemical properties of functionalized PMOs and zeolites L; analytic characterization data of alginate and NC hydrogel scaffolds; SEM images of NC hydrogel scaffolds; quantitative amount of adsorbed proteins on the NC hydrogels; fluorescence microscopy image of cells in the NC hydrogels (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Nermin Seda Kehr: 0000-0003-2275-1254 Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS We thank Deutsche Forschungsgemeinschaft (KE 1577/7-1) for funding, Prof. Harald Fuchs and Prof. Joachim Jose for scientific support, Dr. Celeste Riley Brennecka for corrections in grammar and spelling, and Dr. Azadeh Motealleh for helping us to determine the pore size of the respective scaffolds.
■
REFERENCES
(1) Langer, R.; Vacanti, J. P. Tissue engineering. Science 1993, 260, 920−926. (2) Khademhosseini, A.; Vacanti, J. P.; Langer, R. Progress in Tissue Engineering. Sci. Am. 2009, 300, 64−71. (3) Haraguchi, K. Nanocomposite Hydrogels. Curr. Opin. Solid State Mater. Sci. 2007, 11, 47−57. (4) Schexnailder, P.; Schmidt, G. Nanocomposite Polymer Hydrogels. Colloid Polym. Sci. 2009, 287, 1−11. (5) Wang, C.; Flynn, N. T.; Langer, R. Controlled Structure and Properties of Thermoresponsive Nanoparticle−Hydrogel Composites. Adv. Mater. 2004, 16, 1074−1079. (6) Peak, C. W.; Carrow, J. K.; Thakur, A.; Singh, A.; Gaharwar, A. K. Elastomeric Cell-laden Nanocomposite Microfibers for Engineering Complex Tissues. Cell. Mol. Bioeng. 2015, 8, 404−415. 33681
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
Research Article
ACS Applied Materials & Interfaces (27) Melde, B. J.; Holland, B. T.; Blanford, C. F.; Stein, A. Mesoporous Sieves with Unified Hybrid Inorganic/Organic Frameworks. Chem. Mater. 1999, 11, 3302−3308. (28) Asefa, T.; MacLachlan, M. J.; Coombs, N.; Ozin, G. A. Periodic Mesoporous Organosilicas with Organic Groups Inside the Channel Walls. Nature 1999, 402, 867−871. (29) Ruiz, A. Z.; Bruhwiler, D.; Ban, T.; Calzaferri, G. Synthesis of Zeolite L. Tuning Size and Morphology. Monatsh. Chem. 2005, 136, 77−89. (30) Kehr, N. S.; Ergün, B.; Lülf, H.; De Cola, L. Spatially Controlled Channel Entrances Functionalization of Zeolites L. Adv. Mater. 2014, 26, 3248−3252. (31) Kehr, N. S.; Motealleh, A.; Schäfer, A. H. Cell Growth on (“Janus”) Density Gradients of Bifunctional Zeolite L Crystals. ACS Appl. Mater. Interfaces 2016, 8, 35081−35090. (32) Ergün, B.; De Cola, L.; Galla, H.-J.; Kehr, N. S. SurfaceMediated Stimuli Responsive Delivery of Organic Molecules from Porous Carriers to Adhered Cells. Adv. Healthcare Mater. 2016, 5, 1588−1592. (33) Liberio, M. S.; Sadowski, M. C.; Soekmadji, C.; Davis, R. A.; Nelson, C. C. Differential Effects of Tissue Culture Coating Substrates on Prostate Cancer Cell Adherence, Morphology and Behavior. PLoS One 2014, 9, e112122. (34) Lülf, H.; Bertucci, A.; Septiadi, D.; Corradini, R.; De Cola, L. Multifunctional Inorganic Nanocontainers for DNA and Drug Delivery into Living Cells. Chem. - Eur. J. 2014, 20, 10900−10904. (35) Calzaferri, G.; Huber, S.; Maas, H.; Minkowski, C. Host−Guest Antenna Materials. Angew. Chem., Int. Ed. 2003, 42, 3732−3758. (36) Ko, K. S.; Arora, P. D.; McCulloch, C. A. G. Cadherins Mediate Intercellular Mechanical Signaling in Fibroblasts by Activation of Stretch-sensitive Calcium-permeable Channels. J. Biol. Chem. 2001, 276, 35967−35977. (37) Bershadsky, A. D.; Balaban, N. Q.; Geiger, B. Adhesiondependent Cell Mechanosensitivity. Annu. Rev. Cell Dev. Biol. 2003, 19, 677−695. (38) Lima, W. C.; Vinet, A.; Pieters, J.; Cosson, P. Role of PKD2 in Rheotaxis in Dictyostelium. PLoS One 2014, 9, e88682. (39) Cao, B.; Peng, Y.; Liu, X.; Ding, J. Effects of Functional Groups of Materials on Nonspecific Adhesion and Chondrogenic Induction of Mesenchymal Stem Cells on Free and Micropatterned Surfaces. ACS Appl. Mater. Interfaces 2017, 9, 23574−23585. (40) Lee, S. K.; Han, M. S.; Tung, C.-H. Layered Nanoprobe for Long-Lasting Fluorescent Cell Label. Small 2012, 8, 3315−3320. (41) Lee, S. K.; Mortensen, L. J.; Lin, C. P.; Tung, C.-H. An Authentic Imaging Probe to Track Cell Fate from Beginning to End. Nat. Commun. 2014, 5, 5216. (42) Sun, J.; Graeter, S. V.; Yu, L.; Duan, S.; Spatz, J. P.; Ding, J. Technique of Surface Modification of a Cell-Adhesion-Resistant Hydrogel by a Cell-Adhesion-Available Inorganic Microarray. Biomacromolecules 2008, 9, 2569−2572. (43) Yao, X.; Peng, R.; Ding, J. Cell−Material Interactions Revealed Via Material Techniques of Surface Patterning. Adv. Mater. 2013, 25, 5257−5286. (44) Yan, C.; Sun, J. G.; Ding, J. D. Critical Areas of Cell Adhesion on Micropatterned Surfaces. Biomaterials 2011, 32, 3931−3938.
33682
DOI: 10.1021/acsami.7b10871 ACS Appl. Mater. Interfaces 2017, 9, 33674−33682
