Synthesis and Characterization of Dual Stimuli-Sensitive

Research Article Cite This: ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

www.acsami.org

Synthesis and Characterization of Dual Stimuli-Sensitive Biodegradable Polyurethane Soft Hydrogels for 3D Cell-Laden Bioprinting Shih-Hsiang Hsiao† and Shan-hui Hsu*,†,‡ †

Downloaded via UNIV PIERRE ET MARIE CURIE on August 23, 2018 at 10:14:51 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

Institute of Polymer Science and Engineering, National Taiwan University, Number 1 Section 4 Roosevelt Road, Taipei, 10617 Taiwan, Republic of China ‡ Institute of Cellular and System Medicine, National Health Research Institutes, Number 35 Keyan Road, Miaoli, 35053 Taiwan, Republic of China S Supporting Information *

ABSTRACT: Three-dimensional bioprinting serves as an attractive platform to fabricate customized tissue-engineered substitutes from biomaterials and cells for the repair or replacement of injured tissues and organs. A common challenge for 3D bioprinting materials is that the structures printed from the biodegradable polymer hydrogels tend to collapse because of the poor mechanical stability. In this study, dual stimuli-responsive biodegradable polyurethane (PU) dispersions (PUA2 and PUA3) were synthesized from an eco-friendly waterborne process. Acrylate group was introduced in the PU chain end to serve as a photosensitive moiety for UV-induced cross-linking and improvement of the printability, while mixed oligodiols in the soft segment remained to be the thermosensitive moiety. The photo/thermal-induced morphological changes of PU nanoparticles were verified by dynamic light scattering, small-angle X-ray scattering, and rheological measurement of the dispersions. It was observed that these PU nanoparticles became more rod-like in shape after UV treatment and formed compact packing structures upon further heating. With the thermosensitive properties, these UV-cured PU dispersions underwent rapid thermal gelation with gel moduli in the range 0.5−2 kPa near body temperature. The rheological properties of the PU hydrogels including dynamic viscoelasticity, creep recovery, and shear thinning behavior at 37 °C were favorable for processing by microextrusion-based 3D printing and could be easily mixed with cells before printing to produce cell-laden constructs. The dual-responsive hydrogel constructs demonstrated higher resolution and shape fidelity as well as better cell viability and proliferation than the thermoresponsive control. Moreover, the softer hydrogel (PUA3) with a low modulus ( PUA3 ≫ PUA1. The TEM images of PUA2 and PUA3 NPs without and with UV curing are shown in Figure S1. The sizes of all PU NPs were in the range from 30 to 40 nm at 25 °C. There was a slight size increase at 37 °C. The UV curing resulted in a more rod-like shape and a larger particle size for the NPs at 25 °C. The average sizes obtained from TEM image analyses of the short and long axes were in the range from ∼30 to 55 nm for the cured PUA2 NPs and PUA3 NPs. These TEM sizes were consistent with those obtained from DLS. 3.4. Small-Angle X-ray Scattering (SAXS) Analysis for the PU NP Dispersions. The SAXS scattering intensities of uncured and cured PU NP dispersions at 25 and 37 °C are displayed in Figure 3. The values of Rg and shape factor (Rg/ Rh) obtained from the Guinier analysis are summarized in Table 3. As the temperature was raised from 25 to 37 °C, the F

DOI: 10.1021/acsami.8b08362 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces

Figure 4. Schematics showing the possible mechanism for the dual stimuli-sensitive PU NPs (suspended in aqueous medium) in response to UV treatment and subsequent heating.

Figure 5. Rheological properties of (A) PUA2 and (B) PUA3 dispersions as a function of time (time sweep) in culture medium with UV curing (abbreviated UVm) after placing at 37 °C. Dashed line box in A and B shows the appropriate printing window for 3D printing. (C) Dynamic rheological properties of angular frequency dependence (frequency sweep) at an oscillatory strain (γ = 1%) for the two PU hydrogels. Filled symbols represent storage modulus (G′), and open symbols represent loss modulus (G″). (D) Static shear viscosities of the two PU hydrogels versus shear rate. In C and D, the measurement was conducted after being equilibrated at 37 °C for approximately 1200 s. Solid content of all hydrogels was 25 wt % PU in culture medium. Time sweep tests (A and B) were performed at 1 Hz frequency and 1% oscillatory strain.

Rg of PUA2 increased from 21 to 22.1 nm and that of PUA3 changed from 17.2 to 17.9 nm. No significant Rg change was observed for PUA1 NPs. The UV curing resulted in an increase of Rg (25 °C) for cured PUA2 and PUA3. For cured PUs, the Rg increase at 37 °C (compared to 25 °C) was not so obvious. Among all PU NPs, the cured PUA2 NPs and cured PUA3 NPs at 25 °C displayed larger shape factors at 1.12 and 1.08, respectively (i.e., more rod-like). The shape factors for all uncured PUs and cured PUA1 NPs were below or close to 1 at 25 or 37 °C (i.e., more spherical).

In addition, the apparent molecular weight of PU NPs (Mw,NP) estimated based on Kratky analysis is included in Table 3. The Mw,NP values of uncured PUA1, PUA2, and PUA3 were 49 × 103, 25 × 103, and 25 × 103 kDa, respectively. The Mw,NP values of cured PUA1, PUA2, and PUA3 NPs were 48 × 103, 32 × 103, and 30 × 103 kDa, respectively. Judging from the increased values of Mw,NP upon UV exposure, chemical cross-linking may have occurred among the NPs in PUA2 or PUA3 but not in PUA1 NPs. The values of Nagg obtained from dividing Mw,NP by the molecular weight of a G


Research Article


Figure 6. XRD profiles of (A) PUA1, (B) PUA2, and (C) PUA3 films with and without UV curing (300 s) in the solid state. (D) Degree of chemical cross-linking for PU films, represented by gel fraction (%).

s), while cured PUA2 had a similar thermal gelling profile whether in culture medium or in water. Macroscopic examination (Figure S3) also demonstrated the phenomena of thermally induced gelling for UV-cured PUA2 and PUA3. In the dynamic time sweep experiment, the storage moduli G′ of PUA2 and PUA3 (with UV curing and in the culture medium) were stabilized at about 1.8 and 0.7 kPa after 30 min, respectively. The rheological properties suitable for printing PUA2 and PUA3 hydrogels were after approximately 20 min (printing window at 37 °C) where G′ was about 1.5 and 0.6 kPa for PUA2 and PUA3, respectively. At this time, the tan δ (G″/G′) values were in the range from 0.28 to 0.31. When tan δ was greater than 0.31, the hydrogel would be too weak to be stacked. The optimal modulus and tan δ values allowed the cured PUA2 or PUA3 dispersion to be printed while undergoing thermal gelation and to rapidly recover the solidlike structure to create a stable construct. Figure 5C presents the plots of moduli G′ and G″ versus the oscillatory frequency for PUA2 and PUA3 hydrogels after equilibration at 37 °C for approximately 20 min. The data revealed almost parallel frequency sweep plots of G′ and G″ on a log−log scale. The slope of these plots was 0.179 for PUA2 hydrogel and 0.163 for PUA3 hydrogel. In the literature, the parallel frequency plots of G′ and G″ with G′ greater than G″ in all frequencies indicated a weak gel with shear thinning behavior.42 Many biological fluids such as bronchial or pharyngeal mucus that require fast structure deconstruction and reconstruction to maintain the airway patency have such rheological behavior.43 The shear thinning behavior was confirmed by the steady shear experiment shown in Figure 5D. The steady state viscosities decreased almost linearly with an increase of the shear rate on the log−log scale, indicating the strong shear thinning characteristics of PUA2 and PUA3 hydrogels at this stage.

single chain are listed in Table 3. Each of the uncured PU NPs was a multichain polymer aggregate.41 The NPs of PUA2 and PUA3 had a smaller Nagg value (∼800) than those of PUA1 (∼1600). The interparticle distance among PU NPs could be determined by SAXS profiles (Figure 3). UV curing shortened the distance among the NPs in PUA2 and in PUA3. The spacing was 56.1 nm for both systems. On the other hand, the interparticle distance of PUA1 was beyond the possible range for SAXS observation. On the basis of the above physicochemical characterization, the hypothetical mechanism accounting for the morphological changes of PU NPs upon UV curing and subsequent heating is presented in Figure 4. PUA2 NPs and PUA3 NPs showed photoresponsiveness, which gave rise to increases of Mw,NP and size after UV treatment. Furthermore, the cured PUA2 or PUA3 NPs became more rod-like at higher temperatures. On the other hand, PUA1 NPs did not show any of the above stimuli responsiveness. 3.5. Rheological Properties of PU NP Dispersions. The rheological properties of PU dispersions (in pure water) without and with UV curing were measured after placing the samples at 37 °C, and data are shown in Figure S2. Upon the temperature rise to 37 °C, the moduli G′ and G″ of PUA1 dispersion (30 wt %) did not increase considerably with time. In contrast, uncured PUA2 and PUA3 dispersions underwent a slow sol−gel transition upon moving to 37 °C. Meanwhile, the UV-cured PUA2 dispersion revealed immediate gel-like properties (G′ > G″) with both moduli increased with time when placed at 37 °C. The UV-cured PUA3 dispersion placed at 37 °C underwent sol−gel gelation with G′−G″ crossover in 900 s. The rheological properties of cured PUA2 and PUA3 dispersions prepared in cell culture medium are shown in Figures 5A and 5B. The gelation time of cured PUA3 dispersion was obviously shortened in culture medium (∼317 H


Research Article


Figure 7. Creep and creep recovery strain−time curves for (A) PUA2 and (B) PUA3 hydrogels in the culture medium with UV curing (abbreviated UVm) under various stress levels. Measurement was conducted after being equilibrated at 37 °C for approximately 1200 s. (C) Appearance of the constructs of PUA3 hydrogel fabricated by a 3D bioprinter using two different types of deposition paths (cross-shaped and star-shaped deposition). Images in D indicated that the printed construct of PU hydrogel could be held by hand. Supplemental videos show the printing process (Video S1) and gross appearance (Video S2) of the printed PU construct using PUA3 as an example.

3.6. DSC, XRD, ATR-FTIR, and Gel Fraction. The glass transition temperature (Tg) of PUs without and with UV curing could be obtained from DSC, and the results are summarized in Table 1. PUA3 had the lowest Tg (−61.25 °C), followed by PUA2 (−60.41 °C) and PUA1 (−59.7 °C). The Tg values of PU increased with UV treatment. The extent of microphase separation for cured PUs, judging from the values of Tg,44 was in the order of PUA3 (−58.06 °C) > PUA2 (−57.30 °C) > PUA1 (−57.08 °C). The XRD patterns for PUs in the solid state without and with UV curing are displayed in Figure 6A−C and Table S1. PUA1 showed the sharp crystalline peaks of PCL (2θ = 21.1° and 23.5°). Both PUA2 and PUA3 exhibited two diffraction peaks of PCL, and the former displayed a small diffraction peak at 16.5° attributed to PLLA crystallinity (4.15%). As summarized in Table S1, the degree of crystallinity was the greatest for PUA1 (∼22%). The degree of crystallinity was smaller for PUA2 and PUA3 (∼17%). All PUs after UV curing showed a significant reduction of the crystalline fraction.

The ATR-FTIR was used to examine the changes of hydrogen bonding in different PUs without and with UV curing (solid samples). Spectra in Figure S4 demonstrated that the relative absorption for the peaks at 1730 and 1670 cm−1, each associated with the free carbonyl group and the hydrogenbonded carbonyl group (CO) of PU, changed after UV curing. The relative proportion of hydrogen-bonded CO to total CO was increased by UV treatment for PUA2 and PUA3 but remained similar for PUA1. The increase of hydrogen bonding in PUA2 was greater than that in PUA3 after UV treatment. On the other hand, the cross-linking effect of UV treatment on PU, examined by the gel fraction, is shown in Figure 6D. PUA1, PUA2, and PUA3 after UV curing had a gel fraction of 30−40%. Although some acrylate groups remained unreacted (Figure 2C), the gel fraction was high enough to form stable NPs to support the hydrogel structure for PUA2 and PUA3. 3.7. Creep Resistance and 3D Printing of Hydrogel Constructs. Figure S5 shows the shear strain dependence of G′ and G″ of PU hydrogels obtained from the dynamic I


Research Article

ACS Applied Materials & Interfaces viscoelasticity measurements. When the oscillatory shear strain was over 10%, the G′ value dropped rapidly for PUA2 and PUA3 hydrogels (UV cured). This drop indicated the collapse of gel structure into a quasi liquid state. On the basis of the strain sweep data, we performed creep and creep recovery tests of cured PU hydrogels. Data are shown in Figure 7A and 7B. The creep and creep recovery profiles of PUA2 and PUA3 hydrogels showed viscoelastic−viscoplastic characteristics for 10 cycles. PUA2 hydrogel could resist the creep cycles for a maximum shear stress of 30 Pa and remained stable, while PUA3 hydrogel could resist those of 20 Pa. The flow phenomena occurred at >50 Pa for PUA2 hydrogel and 25 Pa for PUA3 hydrogel. This result was consistent with the dynamic strain sweep data in Figure S5, both indicating that PUA2 hydrogel had a broader range of linear viscoelasticity. The creep recovery also suggested that the solid-like structure of the hydrogel was able to “resume” rapidly after shear removal and was strong enough to resist the loading within the appropriate shear force levels. Using the two-step stimulus condition (Figure S3), PUA2 and PUA3 could be loaded, UV cured, and heated for 3D bioprinting. By tuning the UV curing time and heating time, the PU hydrogels as bioink could be continuously printed through a 210 mm nozzle to produce tissue constructs with the designed dimension of 10 mm × 10 mm × 5 mm (W × D × H), as shown in Figure 7C and Figure S6, in two patterns of fiber stacking (cross shaped or star shaped). After 3D printing, the actual dimensions of the cross-shaped and star-shaped constructs were 9.52 × 9.5 × 4.8 mm3 and 9.89 × 9.76 × 4.95 mm3 for PUA3 and 9.5 × 9.65 × 4.85 mm3 and 10.05 × 10.1 × 4.95 mm3 for PUA2, respectively. The fibers could be stacked up for 25 layers. The diameter of each stacking fiber for the two PUs was ∼250 μm. The printing process of the PU hydrogel is shown in Video S1. Furthermore, the printed PU hydrogel constructs could be picked up, held, and put down by hand, as shown in Figure 7D and Video S2, using the softer hydrogel PUA3 as an example. The weight loss of PUA2 and PUA3 constructs in 37 °C PBS with time is illustrated in Figure S7. The initial weight of the constructs was defined as 100%. Notable weight loss was observed for PU2 (∼10%) and PU3 (∼14%) in 28 days. Although cross-linking may hinder the degradation, PUA2 and PUA3 constructs were gradually degraded by hydrolysis. 3.8. Cell Viability and Proliferation in the Hydrogel Constructs. The viability of fibroblasts in PU hydrogels evaluated by VB-48 is shown in Figure 8A. The immediate cell viability in the culture medium (control), PUA1, PUA2, and PUA3 groups were 71.0%, 62.4%, 74.3%, and 72.8%, respectively. No obvious difference in the immediate cell viability was observed among the groups of culture medium, PUA2, and PUA3. To assess if the printed hydrogel constructs could support long-term cell survival, the cell growth was measured over a 2-week period in culture. The data are shown in Figure 8B. Cells before 3D printing served as the control (100%). After printing, the cell viability decreased to 72% and 77% in PUA2 and PUA3 hydrogel constructs, respectively. In PUA2 constructs, the cell viability increased to 95% after 1 day but decreased again from 1 to 3 days and slightly increased from 3 to 14 days (∼70%). In PUA3 constructs, the cell viability increased significantly after culture and was ∼165% after 7 days, which was an over 2-fold increase compared to the immediate value after printing.

Figure 8. Cell survival and proliferation in the printed hydrogel constructs. (A) Viability of fibroblasts embedded in various hydrogels determined by the VB-48 assay. Control: data obtained from cells in the culture medium. (B) Proliferation of fibroblasts in the gel determined by the CCK-8 assay. Control: data obtained from cells in the gel before 3D printing. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, among the indicated groups.

3.9. Growth and Differentiation of Neural Stem Cells (NSCs) in the Printed PUA3 Hydrogel Constructs. On the basis of the rheological studies mentioned above, PUA3 was a soft and printable hydrogel. The stiffness of PUA3 hydrogel (∼0.7 kPa) was close to that of neural tissues (0.1−10 kPa).45 We further examined the growth and differentiation of NSCs in printed PUA3 hydrogel constructs and compared to those in printed thermosensitive PU hydrogel constructs previously reported, which contained the same amount of PDLLA soft segment (“DL20”) but without photosensitivity.40 The initial cell viability of NSCs in the hydrogel was confirmed (Figure S8), with findings consistent with those in fibroblasts (Figure 8A). The long-term cell growth was measured over a 2-week period in culture, and the result is shown in Figure 9A. After printing, the viability of NSCs decreased to 81% and 67% in PUA3 and DL20 hydrogel constructs, respectively. The healthy cells in gels may start to grow in 3 days of culture. The viability of NSCs in PUA3 and DL20 hydrogel constructs increased significantly after 7 days and were 179% and 154%, J


Research Article


Figure 9. Behavior of NSCs in the printed hydrogel constructs. (A) Cell proliferation in the gel determined by the CCK-8 assay. Control: data obtained from cells in the gel before 3D printing. (B) Fluorescence images of NSCs embedded in PUA3 constructs during a period of 7 days. At 7 days, the morphology of NSCs inside the constructs was also investigated by confocal microscopy. Cells were labeled with PKH26 (red fluorescence). White arrows indicate cell clustering. (C) Expressions of specific neural-related genes (nestin, GFAP, β-tubulin, and MAP2) determined by real-time RT-PCR over a 2-week period in culture. Gene expression was normalized to the housekeeping gene (GAPDH) and represented by the relative ratio of gene expression. Control: data obtained from cells in the tissue culture polystyrene (TCPS). * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, among the indicated groups. DL20: control thermoresponsive PU hydrogel without the HEMA photoresponsive group. K


Research Article


segment in PUA3 compared to PLLA segment in PUA2 presented more steric hindrance and had shorter soft segment length, which may cause a larger free volume and lower thermosensitivity of PUA3 NPs. However, the cross-linking network induced by UV may restrict the mobility of the molecular chain and decrease the free volume in a PU NP, reducing the swelling of cured NPs after further heating. Meanwhile, the electrolytes in the culture medium may produce an electron shielding effect, which reduces the repulsive force on the particle surface and facilitates gelation.48 On the basis of the literature,25 NPs with a greater free volume were more sensitive to electrolytes, and this may explain the larger NP swelling of PUA3 in the culture medium. In our system, there was no macroscopic phase change occurring for PU dispersions exposed to UV for 30 min at room temperature (Figure S3). It was presumed that the presence of water may have decreased the UV curing effect of the PU dispersion (with a water content of 70%). Even so, the photopolymerization of PUs may be nearly completed in 5 min based on ATR-FTIR data. The mechanism behind the photoresponsiveness of PU NPs was elucidated by SAXS analyses. The less compact NP structure of PUA2 and PUA3 could disturb the distribution of the −COOH group on the NP surface, in favor of more extensive cross-linking by free radicals.44,49 Therefore, chemical cross-linking induced by UV may occur simultaneously among NPs, causing increases of size and Mw,NP. In contrast, the distance between PUA1 NPs may be too long for chemical interactions to occur based on the SAXS profile. Among all of the PU NPs, the greater shape factors (Rg/Rh) were found in cured PUA2 and PUA3 NPs, indicating a more rod-like structure. The TEM images revealed that uncured PUA2 and PUA3 were more spherical in shape but transformed into rod-like after UV exposure. Such shape change may come from coalescence of a small portion of NPs. The thermal-induced gelation behavior is that particles attract with each other until they collide to form aggregation. The factors that influence the thermo-sensitive behavior of PU to undergo sol−gel phase transition include the interaction among neighboring NPs (e.g., packing density) and the secondary force interaction (e.g., hydrogen bond).25,27 According to the rheological measurement (time sweep experiment), the cured PU dispersions had a greater tendency to form gels than uncured ones at 37 °C, i.e., shorter gelation time as well as higher gel modulus, in particular for the more thermo-sensitive PUA2. Cured NPs are more rod-like and can be more readily packed than spherical uncured NPs, leading to gel formation.50 In addition, ATR-FTIR results revealed that PUA2 and PUA3 after UV curing had more secondary force interaction, which was reported to facilitate gelation.27 Therefore, the shorter distance among the cured PU NPs arising from size swelling and shape change may enhance the secondary force interaction to undergo phase transition at higher temperatures. Meanwhile, XRD patterns of all PUs synthesized had crystalline peaks, but the peaks were reduced with UV treatment, probably because the cross-linking network formation could restrict PU crystallization. Although most studies considered the ordered structure in soft segment was highly related to thermal gelation, the soft segment crystallinity of PU was not the main factor to influence thermo-sensitivity in the system.25,27 On the basis of results of the above investigations, we developed a new photo/thermal curing system of PU hydrogels (PUA2 and PUA3) that was prepared

respectively, but did not further increase from 10 to 14 days. Fluorescence images of NSCs in PUA3 (Figure 9B) and DL20 (Figure S9) hydrogels revealed that NSCs in both constructs were spherical in shape, and the cell number increased in 7 days. A part of NSCs inside PUA3 hydrogels formed neurosphere-like clusters after 7 days, while cell clustering was not so obvious in DL20 hydrogels. This suggested the ability of PUA3 constructs to support cell attachment and clustering. The marker gene expression (i.e., nestin, GFAP, β-tubulin, and MAP2) for NSCs in the printed constructs after 14 days is displayed in Figure 9C. Cells on the TCPS served as the control. The expression level of nestin gene at 14 days was significantly decreased (∼0.01 and ∼0.25 fold) for NSCs in PUA3 and DL20 hydrogels as compared with the TCPS control, respectively. In contrast, the expression level of GFAP (glial marker), β-tubulin (early neuronal marker), and MAP2 (mature neuronal marker) genes at 14 days was significantly upregulated for NSCs in PUA3 and DL20 hydrogels as compared to those in TCPS control. In particular, NSCs embedded in PUA3 hydrogels expressed the greater levels of GFAP, β-tubulin, and MAP2 genes than those in DL20. To further confirm the differentiation of NSCs inside the constructs, the protein expressions (neural-related biomarkers) after 14 days were performed by immunofluorescent staining, and data are displayed in Figure S10. It was observed that the GFAP, β-tubulin, and MAP2 proteins for NSCs in PUA3 and DL20 were obvious (red fluorescence) but not the nestin protein. The intensity of differentiated marker proteins in PUA3 groups was much greater than those in DL20. These results were consistent with those obtained based on gene expression in Figure 9C.

4. DISCUSSION Three-dimensional bioprinting remains a challenging technology because of the necessity for high structural integrity and fidelity. It is rather difficult to prepare hydrogel ink that has proper mechanical properties while promoting cell proliferation and tissue formation. In this study, we employed a waterborne synthetic route to prepare biodegradable PUs with dual stimuli-responsive properties from a combination of thermosensitive mixed oligodiols and photosensitive acrylate groups.46 This combination took advantage of the UV curing process to enhance the cell printing possibility and structural stability.47 To synthesize such dual-responsive PU, acrylate groups were partially introduced into the end of the thermosensitive PU main chains with single-hydroxyl acrylate (HEMA) to generate vinyl-terminated PUs, where the acrylic functionality served as the UV curing site.44 Three PU NP dispersions composed of different ratios of oligodiols in the soft segment and same acrylate monomer for chain termination (Table 1) were prepared in this study and compared for their stimuli-responsive behavior and printing ability. The size change of PU NPs in dispersion was related to their water swelling. When the temperature was raised from 25 to 37 °C, the NPs of PUA2 and PUA3 revealed significant swelling but not the NPs of PUA1. According to the literature, the swelling of PU NPs may be attributed to the microphase separation of PU.27 In this study, the greater microphase separation of PUA2 and PUA3 arising from incompatibility between the two different soft segments could lead to greater chain mobility at higher temperatures, which may account for the significant thermal swelling of the NPs. The PDLLA L


Research Article


thinning behavior of materials is likely to result in cell injury during the printing process because of the shear stresses inflicted on cells in viscous fluids.7 Even so, the cell viability in PUA3 hydrogel after printing could be recovered and was an over 2-fold increase after culture for 7 days. Regarding the mechanical properties, the literature has proposed that the matrix of 0.1−10 kPa stiffness may offer a more favorable environment for NSCs.45 Meanwhile, the shear storage modulus of cerebrum white matter tissue from corona radiata was in the range of 0.3−0.5 kPa at a frequency of 1 Hz.57 We further examined the growth and differentiation of NSCs in PUA3 hydrogel (∼0.7 kPa) and compared to previously reported DL20 hydrogel (∼9 kPa) for potential applications in neural tissue engineering.46 The immediate cell viability data demonstrated that NSCs had better survival in PUA3 compared to the DL20 hydrogel (Figure S8). The long-term cell viability in PU hydrogels exhibited almost the same tendency as the immediate cell viability result. NSCs inside PUA3 constructs could form neurosphere-like clusters after culture for 7 days. Marker gene analyses revealed that NSCs embedded in PUA3 and DL20 hydrogels expressed increased levels of GFAP, β-tubulin, and MAP2 genes after culture for 14 days without the induction medium, suggesting that both hydrogels might provide a suitable environment for NSC differentiation toward glial and neuronal lineages. It was noted that NSCs in PUA3 hydrogel exhibited significantly higher expression levels of β-tubulin and MAP2 genes, indicating more neuronal differentiation. Similar tendencies were observed in protein expression. The observation suggested that the softer PUA3 hydrogel may have simulated the mechanical environment of brain tissue.58 Factors that determine the printability and bioactivities of UV-cured PU hydrogels include the chemical composition of soft segment and other reactants, the reactivity of functional groups, and species of the photoinitiator. According to a recent work,22 a further improvement of our current system may be to place the acrylate monomer HEMA in the PU chain end by acryloyl chloride that could be added after the chain extender EDA because of its lower reaction temperature (40 °C). The PU-acrylate generated by this way may have longer molecular chain length and better thermoresponsiveness. Meanwhile, visible light for curing is safer than UV cross-linking and can be considered for the work in the future.22 Taken together, our study revealed the unique photo/thermal-induced gelation mechanism and rheological properties of novel biodegradable PU hydrogels as well as the possibility to establish soft (tofulike) bioink formulas that could be mixed with cells and printed with high structural fidelity and stability. The soft and printable hydrogels will contribute toward the custom-made biomanufacturing of soft tissues.

from UV exposure of the PU dispersions for 5 min and subsequent preheating at 37 °C (Figure S3). Rheological properties are believed to play an important role in 3D printability of the hydrogels. Both cured PUA2 and PUA3 hydrogels in the culture medium had weak gel modulus close to or smaller than 1 kPa. Such modulus was much smaller than other thermoprintable hydrogels previously reported.24,25,46 This was probably owing to a lower extent of hydrogen bonding.27 The cured hydrogels could undergo a progressive breakdown into smaller aggregates as frequencies increase and behave similarly to those typical of physically cross-linked networks.51 Therefore, the cured PU hydrogels showed strong shear thinning behavior and could be thinned into liquid form under the high shear rates during printing. Upon deformation and removal of shear stress within the linear viscoelastic region (e.g., after printing), the cured PU hydrogels were capable of quickly recovering back into gel form to maintain their original modulus and filamentary shape but generally incompletely. It has been previously reported that the inherent elasticity and cross-linking degree of a hydrogel may allow them to recover after exiting the nozzle.52,53 This ability to rapidly rebuild the structure was important for keeping the stability of the printed construct. Moreover, the cured PUA2 hydrogel had a higher shear yield stress, indicating that constructs printed from PUA2 were more structurally stable than those printed from PUA3. On the basis of the above analyses, the rheological behaviors of cured hydrogels were similar to those reported for the printable ink.54 Besides, the new gels could offer a rather soft (tofu-like) but stable 3D environment. The extrusion-based printing technologies could be used to create layered 3D structures. The advantage of PUA2 and PUA3 hydrogels as printing ink was its relatively low viscosity during extrusion that could avoid excessive fluid shear stress and potential for jamming. Meanwhile, the structure strength and shear yield stress of our hydrogels were enough for bearing the weight of ink without obviously changing the shape of stacking fibers during the process of bottom-up layer-by-layer deposition. In this study, the cured PU hydrogels were successfully printed in cross-shaped and star-shaped patterns through a 210 mm nozzle at 37 °C. The actual volume of cross-shaped and star-shaped PUA3 constructs after printing was reduced 13.2% (from 10 × 10 × 5 mm3 to 9.52 × 9.5 × 4.8 mm3) and 4.4% (from 10 × 10 × 5 mm3 to 9.89 × 9.76 × 4.95 mm3), respectively, which represented a reasonably good pattern and dimension fidelity. Most impressively, the printed constructs exhibited solid-like behavior so that they could be picked up from the platform by hand, like holding tofu (using PUA3 as an example). To evaluate the tissue engineering applications of our hydrogels, fibroblasts were embedded into the hydrogels for printing at 37 °C and analyzed preliminarily for cell viability and growth. Results showed that the printed PUA3 hydrogel constructs possessed a higher ratio of viable cells than the printed PUA2 hydrogel constructs after 14 days of culture. An earlier study also demonstrated that hydrogel comprising PDLLA segment was more biocompatible than that comprising PLLA segment.24 Several studies have reported that PLLA generated lactic acid during the processes of degradation; the accumulated degradation metabolites deprived the cell growth and provoked inflammation in the surrounding tissues.55,56 This may explain why the cell viability of PUA2 and PUA3 did not increase from 10 to 14 days. On the other hand, the shear

5. CONCLUSIONS Novel waterborne and biodegradable PU NP dispersions with dual stimuli-responsive properties were synthesized and characterized in this study. They were cured by the optimized two-step stimulation through adjusting the UV exposure and preheating time. As revealed by SAXS, the relatively loose structure of NPs in the formulas comprising mixed oligodiols (i.e., the cases of PUA2 and PUA3) may result in more effective UV-induced cross-linking among NPs. Such interparticle cross-linking may increase the secondary force interaction and transform the NPs from spherical to rod-like structure, thereby facilitating gel formation upon further M


Research Article


(3) Xie, Y.; Lan, X. R.; Bao, R. Y.; Lei, Y.; Cao, Z. Q.; Yang, M. B.; Yang, W.; Wang, Y. B. High-Performance Porous Polylactide Stereocomplex Crystallite Scaffolds Prepared by Solution Blending and Salt Leaching. Mater. Sci. Eng., C 2018, 90, 602−609. (4) Ma, T.; Zhang, Y. S.; Chen, A. Z.; Ju, J.; Gu, C. W.; Kankala, R. K.; Wang, S. B. Carbon Dioxide-Assisted Bioassembly of Cell-Loaded Scaffolds From Polymeric Porous Microspheres. J. Supercrit. Fluids 2017, 120, 43−51. (5) Chen, Q. B.; Kankala, R. K.; Chen, A. Z.; Yang, D. Z.; Cheng, X. X.; Jiang, N. N.; Zhu, K.; Wang, S. B. Investigation of Silk Fibroin Nanoparticle-Decorated Poly(L-lactic acid) Composite Scaffolds for Osteoblast Growth and Differentiation. Int. J. Nanomed. 2017, 12, 1877−1890. (6) Kankala, R. K.; Zhang, Y. S.; Wang, S. B.; Lee, C. H.; Chen, A. Z. Supercritical Fluid Technology: An Emphasis on Drug Delivery and Related Biomedical Applications. Adv. Healthcare Mater. 2017, 6, 1700433. (7) Murphy, S. V.; Atala, A. 3D bioprinting of Tissues and Organs. Nat. Biotechnol. 2014, 32, 773−785. (8) Kankala, R. K.; Zhu, K.; Sun, X. N.; Liu, C. G.; Wang, S. B.; Chen, A. Z. Cardiac Tissue Engineering on the Nanoscale. ACS Biomater. Sci. Eng. 2018, 4 (3), 800−818. (9) Drury, J. L.; Mooney, D. J. Hydrogels for Tissue Engineering: Scaffold Design Variables. Biomaterials 2003, 24, 4337−4351. (10) Hoffman, A. S. Hydrogels for Biomedical Applications. Adv. Drug Delivery Rev. 2012, 64, 18−23. (11) Chang, R.; Nam, J.; Sun, W. Direct Cell Writing of 3D Microorgan for In Vitro Pharmacokinetic Model. Tissue Eng., Part C 2008, 14, 157−166. (12) Dolati, F.; Yu, Y.; Zhang, Y.; Jesus, A. M. D.; Sander, E. A.; Ozbolat, I. T. In Vitro Evaluation of Carbon-Nanotube-Reinforced Bioprintable Vascular Conduits. Nanotechnology 2014, 25, 145101. (13) England, S.; Rajaram, A.; Schreyer, D. J.; Chen, X. Bioprinted Fibrin-Factor XIII-Hyaluronate Hydrogel Scaffolds with Encapsulated Schwann Cells and Their In Vitro Characterization for Use in Nerve Regeneration. Bioprinting 2017, 5, 1−9. (14) Tan, Y. J.; Tan, X.; Yeong, W. Y.; Tor, S. B. Hybrid Microscaffold-Based 3D Bioprinting of Multi-Cellular Constructs with High Compressive Strength: A New Biofabrication Strategy. Sci. Rep. 2016, 6, 39140. (15) Murphy, M.; Kolan, K.; Li, W.; Semon, J.; Day, D.; Leu, M. 3D Bioprinting of Stem Cells and Polymer/Bioactive Glass Composite Scaffolds for Bone Tissue Engineering. Int. J. Bioprint. 2017, 3, 1−11. (16) Wüst, S.; Godla, M. E.; Müller, R.; Hofmann, S. Tunable Hydrogel Composite with Two-Step Processing in Combination with Innovative Hardware Upgrade for Cell-Based Three-Dimensional Bioprinting. Acta Biomater. 2014, 10, 630−640. (17) Ouyang, L.; Highley, C. B.; Sun, W.; Burdick, J. A. A Generalizable Strategy for the 3D Bioprinting of Hydrogels from Nonviscous Photo-crosslinkable Inks. Adv. Mater. 2017, 29, 1604983. (18) Du, Y.; Lo, E.; Ali, S.; Khademhosseini, A. Directed Assembly of Cell-Laden Microgels for Fabrication of 3D Tissue Constructs. Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 9522−9527. (19) Bertassoni, L. E.; Cardoso, J. C.; Manoharan, V.; Cristino, A. L.; Bhise, N. S.; Araujo, W. A.; Zorlutuna, P.; Vrana, N. E.; Ghaemmaghami, A. M.; Dokmeci, M. R.; Khademhosseini, A. Direct-Write Bioprinting of Cell-Laden Methacrylated Gelatin Hydrogels. Biofabrication 2014, 6, 024105. (20) Colosi, C.; Shin, S. R.; Manoharan, V.; Massa, S.; Costantini, M.; Barbetta, A.; Dokmeci, M. R.; Dentini, M.; Khademhosseini, A. Microfluidic Bioprinting of Heterogeneous 3D Tissue Constructs Using Low-Viscosity Bioink. Adv. Mater. 2016, 28, 677−684. (21) Bertassoni, L. E.; Cecconi, M.; Manoharan, V.; Nikkhah, M.; Hjortnaes, J.; Cristino, A. L.; Barabaschi, G.; Demarchi, D.; Dokmeci, M. R.; Yang, Y.; Khademhosseini, A. Hydrogel Bioprinted Microchannel Networks for Vascularization of Tissue Engineering Constructs. Lab Chip 2014, 14, 2202−2211.

heating. ATR-FTIR confirmed the greater extent of hydrogen bonding in cured PUA2 and PUA3. Cured PU hydrogels at 37 °C exhibited solid-like (low tan δ) and shear thinning behavior as well as creep recovery for the sheared low-viscosity materials to rebuild the hydrogel structure quickly, allowing them to be 3D printed with cells by a continuous multilayer deposition process. The printed hydrogels were relatively soft (tofu-like) but with structural stability and dimension fidelity. After printing, cells embedded in PUA3 hydrogels demonstrated fast proliferation in 1 week. Moreover, the stiffness of PUA3 hydrogel constructs (∼0.7 kPa) was conducive to the survival and proliferation of NSCs as well as their differentiation into neural cells. The cured PUA3 tofu-like hydrogel is a promising candidate ink for use in 3D bioprinting of soft tissues, particularly in the field of neural tissue engineering or fabrication of simulated brains in the future.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.8b08362. Crystallinity and thermal properties, TEM images for the morphology of PU NPs, rheological profiles of PU dispersions by time sweep and strain sweep tests, macroscopic observation for the phase states of PU hydrogels and constructs, ATR-FTIR spectra, in vitro degradation test, cell viability, cell images, and protein expression of NSCs embedded in hydrogels (PDF) Video showing the printing process of the printed PU hydrogel construct (AVI) Video showing the gross appearance of the printed PU hydrogel construct (AVI)

■

AUTHOR INFORMATION

Corresponding Author

*Phone: +886-2-3366-5313; Fax: +886-2-3366-5237; E-mail: [email protected]. ORCID

Shan-hui Hsu: 0000-0003-3399-055X Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was supported by the Program for Additive Manufacturing (MOST 106-3114-E-002-019), Ministry of Science and Technology, Taiwan, and partially supported by the institutional resource of National Health Research Institutes (Central Government S&T Grant, Taiwan 1070324-01-19-03). Also, the Joint Center for Instruments and Researches, College of Bioresources and Agriculture, National Taiwan University, is thanked for the assistance of confocal microscopy. We also thank the National Synchrotron Radiation Research Center (2018-1-138) and the staff, particularly, Dr. U.-Ser Jeng, for providing the resources and technical support.

■

REFERENCES

(1) Chia, H. N.; Wu, B. M. Recent Advances in 3D Printing of Biomaterials. J. Biol. Eng. 2015, 9, 4. (2) Kankala, R. K.; Zhu, K.; Li, J.; Wang, C. S.; Wang, S. B.; Chen, A. Z. Fabrication of Arbitrary 3D Components In Cardiac Surgery: From Macro-, Micro- to Nanoscale. Biofabrication 2017, 9, 032002. N


Research Article


3D Bioprinting and Differentiation of Neural Stem Cells. J. Mater. Chem. B 2017, 5, 8854−8864. (41) Zhu, Z.; Xie, C.; Liu, Q.; Zhen, X.; Zheng, X.; Wu, W.; Li, R.; Ding, Y.; Jiang, X.; Liu, B. The Effect of Hydrophilic Chain Length and iRGD on Drug Delivery From Poly(ε-caprolactone)-Poly(Nvinylpyrrolidone). Biomaterials 2011, 32, 9525−9535. (42) Rosalina, I.; Bhattacharya, M. Dynamic Rheological Measurements and Analysis of Starch Gels. Carbohydr. Polym. 2002, 48, 191− 202. (43) Nordgård, C. T.; Draget, K. I. Oligosaccharides as Modulators of Rheology in Complex Mucous Systems. Biomacromolecules 2011, 12, 3084−3090. (44) Wang, F.; Hu, J. Q.; Tu, W. P. Study on Microstructure of UVurable Polyurethane Acrylate films. Prog. Org. Coat. 2008, 62, 245− 250. (45) Saha, K.; Keung, A. J.; Irwin, E. F.; Li, Y.; Little, L.; Schaffer, D. V.; Healy, K. E. Substrate Modulus Directs Neural Stem Cell Behavior. Biophys. J. 2008, 95, 4426−4438. (46) Ho, L.; Hsu, S. h. Cell Reprogramming by 3D Bioprinting of Human Fibroblasts in Polyurethane Hydrogel for Fabrication of Neural-like Constructs. Acta Biomater. 2018, 70, 57−70. (47) Pereira, I. H. L.; Ayres, E.; Patrício, P. S.; Góes, A. M.; Gomide, V. S.; Junior, E. P.; Oréfice, R. L. Photopolymerizable and Injectable Polyurethanes for Biomedical Applications: Synthesis and Biocompatibility. Acta Biomater. 2010, 6, 3056−3066. (48) Dautzenberg, H. Polyelectrolyte Complex Formation in Highly Aggregating Systems. 1. Effect of Salt: Polyelectrolyte Complex Formation in the Presence of NaCl. Macromolecules 1997, 30, 7810− 7815. (49) Yang, S. M.; Kim, S. H.; Lim, J. M.; Yi, G. R. Synthesis and Assembly of Structured Colloidal Particles. J. Mater. Chem. 2008, 18, 2177−2190. (50) Philipse, A. P.; Wierenga, A. M. On the Density and Structure Formation in Gels and Clusters of Colloidal Rods and Fibers. Langmuir 1998, 14, 49−54. (51) Vermonden, T.; Besseling, N. A. M.; van Steenbergen, M. J.; Hennink, W. E. Rheological Studies of Thermosensitive Triblock Copolymer Hydrogels. Langmuir 2006, 22, 10180−10184. (52) Hung, K. C.; Tseng, C. S.; Hsu, S. h. Synthesis and 3D Printing of Biodegradable Polyurethane Elastomer by a Water-Based Process for Cartilage Tissue Engineering Applications. Adv. Healthcare Mater. 2014, 3, 1578−1587. (53) Tanpichai, S.; Oksman, K. Cross-linked Nanocomposite Hydrogels Based On Cellulose Nanocrystals and PVA: Mechanical Properties and Creep Recovery. Composites, Part A 2016, 88, 226− 233. (54) Park, J.; Lee, S. J.; Chung, S.; Lee, J. H.; Kim, W. D.; Lee, J. Y.; Park, S. A. Cell-Laden 3D Bioprinting Hydrogel Matrix Depending on Different Compositions for Soft Tissue Engineering: Characterization and Evaluation. Mater. Sci. Eng., C 2017, 71, 678−684. (55) Guo, Z.; Bo, D.; He, Y.; Luo, X.; Li, H. Degradation Properties of Chitosan Microspheres/Poly(L-lactic acid) Composite In Vitro and In Vivo. Carbohydr. Polym. 2018, 193, 1−8. (56) Feng, G.; Qin, C.; Yi, X.; Xia, J.; Chen, J.; Chen, X.; Chen, T.; Jiang, X. Effect of Novel Bioresorbable Scaffold Composed of Poly-Llactic Acid and Amorphous Calcium Phosphate Nanoparticles on Inflammation and Calcification of Surrounding Tissues After Implantation. J. Mater. Sci.: Mater. Med. 2018, 29, 112. (57) Garo, A.; Hrapko, M.; Dommelen, J. A. W.; Peters, G. W. M. Towards a Reliable Characterisation of the Mechanical Behaviour of Brain Tissue: The Effects of Post-mortem Time and Sample Preparation. Biorheology 2007, 44, 51−58. (58) Lu, Y. B.; Franze, K.; Seifert, G.; Steinhäuser, C.; Kirchhoff, F.; Wolburg, H.; Guck, J.; Janmey, P.; Wei, E. Q.; Käs, J.; Reichenbach, A. Viscoelastic Properties of Individual Glial Cells and Neurons in the CNS. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 17759−17764.

(22) Xu, C.; Lee, W.; Dai, G.; Hong, Y. Highly Elastic Biodegradable Single-Network Hydrogel for Cell Printing. ACS Appl. Mater. Interfaces 2018, 10, 9969−9979. (23) Gunatillake, P. A.; Adhikari, R. Biodegradable Synthetic Polymers for Tissue Engineering. Eur. Cell Mater. 2003, 5, 1−16. (24) Hsieh, F. Y.; Lin, H. H.; Hsu, S. h. 3D Bioprinting of Neural Stem Cell-Laden Thermoresponsive Biodegradable Polyurethane Hydrogel and Potential in Central Nervous System Repair. Biomaterials 2015, 71, 48−57. (25) Lin, H. H.; Hsieh, F. Y.; Tseng, C. S.; Hsu, S. h. Preparation and Characterization of A Biodegradable Polyurethane Hydrogel and The Hybrid Gel with Soy Protein for 3D Cell-Laden Bioprinting. J. Mater. Chem. B 2016, 4, 6694−6705. (26) Lei, M.; Wang, X. Biodegradable Polymers and Stem Cells for Bioprinting. Molecules 2016, 21, 539. (27) Ou, C. W.; Su, C. H.; Jeng, U. S.; Hsu, S. h. Characterization of Biodegradable Polyurethane Nanoparticles and Thermally Induced Self-Assembly in Water Dispersion. ACS Appl. Mater. Interfaces 2014, 6, 5685−5694. (28) Zhou, H.; Yu, B.; Zhou, J.; Zeng, J.; Zhou, Z.; Liu, G. Synthesis and Characterization of Thermal- and pH-Sensitive Polyurethane Hydrogels with Different Transition Temperature. Nanosci. Nanotechnol. Lett. 2016, 8, 647−653. (29) Andriani, Y.; Chua, J. M.; Chua, B. Y.; Phang, I. Y.; ShyhChang, N.; Tan, W. S. Polyurethane Acrylates as Effective Substrates for Sustained In Vitro Culture of Human Myotubes. Acta Biomater. 2017, 57, 115−126. (30) Hanson Shepherd, J. N.; Parker, S. T.; Shepherd, R. F.; Gillette, M. U.; Lewis, J. A.; Nuzzo, R. G. 3D Microperiodic Hydrogel Scaffolds for Robust Neuronal Cultures. Adv. Funct. Mater. 2011, 21, 47−54. (31) Morris, V. B.; Nimbalkar, S.; Younesi, M.; McClellan, P.; Akkus, O. Mechanical Properties, Cytocompatibility and Manufacturability of Chitosan: PEGDA Hybrid-Gel Scaffolds by Stereolithography. Ann. Biomed. Eng. 2017, 45, 286−296. (32) Poldervaart, M. T.; Goversen, B.; de Ruijter, M.; Abbadessa, A.; Melchels, F. P. W.; Ö ner, F. C.; Dhert, W. J. A.; Vermonden, T.; Alblas, J. 3D Bioprinting of Methacrylated Hyaluronic Acid (MeHA) Hydrogel with Intrinsic Osteogenicity. PLoS One 2017, 12, e0177628. (33) Occhetta, P.; Visone, P.; Russo, L.; Cipolla, L.; Moretti, M.; Rasponi, M. VA-086 Methacrylate Gelatine Photopolymerizable Hydrogels: A Parametric Study for Highly Biocompatible 3D Cell Embedding. J. Biomed. Mater. Res., Part A 2015, 103, 2109−2117. (34) Rouillard, A. D.; Berglund, C. M.; Lee, J. Y.; Polacheck, W. J.; Tsui, Y.; Bonassar, L. J.; Kirby, B. J. Methods for Photocrosslinking Alginate Hydrogel Scaffolds with High Cell Viability. Tissue Eng., Part C 2011, 17, 173−179. (35) Billiet, T.; Gevaert, E.; De Schryver, T.; Cornelissen, M.; Dubruel, P. The 3D printing of Gelatin Methacrylamide Cell-Laden Tissue-Engineered Constructs with High Cell Viability. Biomaterials 2014, 35, 49−62. (36) Pyo, S. H.; Wang, P.; Hwang, H. H.; Zhu, W.; Warner, J.; Chen, S. Continuous Optical 3D Printing of Green Aliphatic Polyurethanes. ACS Appl. Mater. Interfaces 2017, 9, 836−844. (37) Shie, M. Y.; Chang, W. C.; Wei, L. J.; Huang, Y. H.; Chen, C. H.; Shih, C. T.; Chen, Y. W.; Shen, Y. F. 3D Printing of Cytocompatible Water-Based Light-Cured Polyurethane with Hyaluronic Acid for Cartilage Tissue Engineering Applications. Materials 2017, 10, 136. (38) Kratky, O. Possibilities of X-ray Small Angle Analysis in The Investigation of Dissolved and Solid High Polymer Substances. Pure Appl. Chem. 1966, 12, 483−523. (39) Hsu, S. h.; Chen, C. W.; Hung, K. C.; Tsai, Y. C.; Li, S. Thermo-Responsive Polyurethane Hydrogels Based on Poly (εcaprolactone) Diol and Amphiphilic Polylactide-Poly (Ethylene Glycol) Block Copolymers. Polymers 2016, 8, 252. (40) Huang, C. T.; Kumar Shrestha, L.; Ariga, K.; Hsu, S. h. A Graphene-Polyurethane Composite Hydrogel as a Potential Bioink for O


Synthesis and Characterization of Dual Stimuli-Sensitive

Recommend Documents