- Email: [email protected]
Three-dimensional printing of shape memory hydrogels with internal structure for drug delivery
Materials Science & Engineering C 84 (2018) 44–51
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Three-dimensional printing of shape memory hydrogels with internal structure for drug delivery
T
Yongzhou Wanga, Ying Miaoa, Jieling Zhanga, Jian Ping Wub, Thomas Brett Kirkb, Jiake Xuc, ⁎ ⁎ Dong Maa, , Wei Xuea, a
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China 3D Imaging and Bioengineering Laboratory, Department of Mechanical Engineering, Curtin University, Perth, Australia c The School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia b
A R T I C L E I N F O
A B S T R A C T
Keywords: 3D printing Shape memory hydrogels Internal structure Rapid drug release
Hydrogels with shape memory behavior and internal structure have wide applications in fields ranging from tissue engineering and medical instruments to drug delivery; however, creating the hydrogels has proven to be extremely challenging. This study presents a three-dimensional (3D) printing technology to fabricate the shape memory hydrogels with internal structure (SMHs) by combining sodium alginate (alginate) and pluronic F127 diacrylate macromer (F127DA). SMHs were constituted by a dual network structure. One is a stable network which is formed by F127DA photo-crosslinking; the other one is a reversible network which is formed by Ca2 + cross-linked alginate. SMHs recovery ratio was 98.15% in 10 min after Ca2 + was removed in the Na2CO3 solution, and the elastic modulus remains essentially stable after the shape memory cycle. It showed that the drug releasing rate is more rapid compared with traditional drug-loaded hydrogels in in vitro experiments. The viability of 3T3 fibroblasts remained intact which revealed its excellent biocompatibility. Therefore, SMHs have a huge prospect for application in drug carriers and tissue engineering scaffold.
1. Introduction Shape memory hydrogels are a class of gel that can restore its original form in the presence of external stimuli [1–3]. Lendlein reported a PCL-based shape memory polymer (SMPs) and demonstrated its potential in medical applications [4]. Since then more and more research focused on the development of SMPs biocompatible applications [5,6]. Willner et al. have developed DNA hydrogels with shape memory performance by regulating pH value [7,8]. Yakacki et al. have proposed a shape memory polymer for cardiovascular stent [9]. Shape-memory in drug controlled release devices was reported by Wischke et al. [10]. Drug release from the tablets depended on the surface area to volume ratio [11]. The internal structure of the hydrogel can change its surface area volume ratio to have a positive effect on drug controlled release [12]. The internal structure of the hydrogel is widely used in cell culture [13] and tissue engineering scaffolds [14,15] because it uses less material to provide space support for cell growth. For example, ErndtMarino et al. reported a cell layer-electrospun mesh internal structure as coronary artery bypass grafts [16]. Lin et al. reported 3D printing polyrotaxane-based lattice cubes which are capable of converting the chemical energy input into mechanical work; it is also proved that the
⁎
polymer without the internal structure cannot achieve this conversion effect [17]. So the shape memory hydrogel with internal structure has a huge potential value in the medical field. But there is still no effective way to control the internal structure of the shape memory hydrogels. To address this issue, an attractive strategy would be the introduction of a 3D printing technique applied to the manufacturing process of shape memory hydrogels. 3D printing is recognized as a promising technology since it was developed by Charles Hull in the early 1980s [18]. Nowadays, the development of computer-aided design and image processing enable 3D printing to reconstruct an accurate physical model [19–21]. 3D printing technology used in the manufacture of SMPs is also gradually reported [17]. A previous work carried out by Zarek et al. described 3D printing of shape memory polymers that can be used in flexible electronic devices [22]. Integrating 3D printability to SMPs will allow us to explore the possibility of 3D printing SMHs. Here, we report the design and synthesis of printable hydrogels (Fig. 1) which are composed of alginate and pluronic F127 (EO100PO65-EO100; PEO = poly (ethylene oxide), PPO = poly (propylene oxide)). F127 is a common material in 3D printing areas and it has a sol–gel transition near physiological temperatures [23,24]. Pluronic F127 diacrylate macromer (F127DA) also has this characteristic
Corresponding authors. E-mail addresses: [email protected] (D. Ma), [email protected] (W. Xue).
https://doi.org/10.1016/j.msec.2017.11.025 Received 31 July 2017; Received in revised form 11 October 2017; Accepted 22 November 2017 Available online 23 November 2017 0928-4931/ © 2017 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 1. The 3D printed shape memory hydrogel illustration. (a), (b), (c) The molecular mechanism of 3D printed shape memory hydrogels. (d) 3D printing memory hydrogel macro performance.
used as received without further purification.
because it retains a PEO-PPO-PEO tri-block copolymer structure [25]. F127DA has been reported for the manufacture of multi-responsive [26] and self-healing [27] hydrogels with excellent mechanical properties. F127DA can be crosslinked under the excitation of a photoinitiator to form a stable hydrogel at room temperature. The hydrogel was printed (printing state) by a 3D printer. The original shape of the hydrogel (original state) was formed by UV cross-linking of F127DA. Alginate as a natural polysaccharide which can form a high viscosity aqueous solution was widely used in 3D printing because of its good biocompatibility [28–30]. We selected alginate as the other material because the chelating of alginate with Ca2 + imparts a shape memory function to hydrogels [31]. Ca2 + cross-linked sodium alginate fixes the temporary shape of the hydrogel (temporary state). The hydrogel was restored to its original shape (recovery state) by Ca2 + substitution which was by destroyed the cross-linked network of calcium alginate. This study explores printing ink and printing parameters to create the excellent shape memory behavior of SMHs. The printing ink was designed by combining F127DA and alginate. The printing parameters were explored by calculating the expansion rate of the print lines. The shear modulus of hydrogels in the shape memory behavior cycle was analyzed by rheological experiments. It is also reported here that SMPs have good biocompatibility and can be rapidly released as a drug carrier.
2.2. F127DA-alginate ink fabrication A critical first step was to develop printing ink, while the F127DA component was the principle determinant of initial print quality. Therefore, it was necessary to first determine the content of component F127DA. The ink in 3D printing compatibility experiment was tested over a range of 10%, 12%, 14%, 16%, 18% and 20% (w/v) F127DA with 4% (w/v) alginate. The shear viscosity of different concentrations of F127DA was measured at 25 °C. Then the ink was printed into lines to determine its expansion rate at different platform temperatures. The line expansion rate is the ratio of the diameter of the print line to the nozzle diameter. The line diameter was measured three times by a micrometer. Alginate-Ca2 + coordination was the key to the shape memory function of the hydrogels. Accordingly, alginate was 2%, 4% and 6% (w/v) in the ink containing an equal amount F127DA, and the ink was used to print and evaluated its shape memory cycles to select the optimum alginate concentration. The quantitative shape memory cycle was determined according to the reported method [32]. Briefly, straight shapes were printed and cured with UV light for 3 min to form a hydrogel. It was bent into a U-form and immersed into 1% (w/v) CaCl2 solution for 2 min. Then the deformed hydrogel was transferred into 2% (w/v) Na2CO3 solution to remove Ca2 + in the hydrogel. The shape memory cycles were evaluated by measuring the angle at specific time points. The shape fixity ratio (Rf) and shape recovery ratio (Rr) were defined by the following equations [33]:
2. Experimental sections 2.1. Materials F127DA modified from F127, which includes material, pluronic F127 (Sigma, USA), acrylo acryloyl chloride (Aladdin, USA), triethylamine and dichloromethane (Tianjin Damao Chemical Reagents Company, China). 2,2-Dimethoxy-2-phenylacetophenone (DMPA) (Acros, USA) was dissolved in 1-vinyl-2-pyrrolidone (VP) (Aladdin, USA) as a photoinitiator. Alginate and methotrexate (MTX) were both purchased from Aladdin. All other chemicals were analytical grade and
Rf = θt /θi ∗100% Rr = (θi − θf )/θi ∗100% While θi is the given angle, θt is the temporarily fixed angle and θf is the final angle. The contents of the two components in the ink were determined by 45
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
the hydrogels, the drug release of 3D printing hydrogel of recovery state was explored. The MTX-loading hydrogels were placed into a vial containing 10 mL PBS (pH = 7.4) at 37 °C. Then 2 mL release media was taken out and 2 mL fresh pre-warmed release media was added back at every predetermined time points. The amount of released MTX was analyzed by UV–Vis spectrophotometer. All release studies were carried out in triplicate.
the above experiment. F127DA was dissolved in deionized water at a low temperature environment to form a uniform solution, followed by adding sodium alginate by slow and even magnetic stirring, and finally adding the photosensitizer solution (20 μL/mL) stirring evenly. F127DA was modified from F127. It was synthesized according to the reported method [34]. Briefly, 3.2 g F127 (dried in vacuum at 40 °C for 12 h) was dissolved in 25 mL dry CH2Cl2 in a 250 mL round bottomed flask and cooled to 0 °C in an ice bath. Then, triethylamine and acryloyl chloride were added dropwise to the solution under continuous magnetic stirring for more than 30 min then stirring at 0 °C and at room temperature for 12 h, respectively. After filtering off the by-product triethylamine hydrochloride, the mixture was washed by an excess of dichloromethane. After drying at 40 °C under vacuum for 24 h, the F127DA was obtained with a yield of 89%. The photosensitizer solution was prepared by 0.1 g DMPA dissolved in 1 mL VP.
2.8. Cytotoxicity 3T3 cells were incubated with the hydrogel for testing the cytotoxicity in this work. For the samples' preparation, the temporary and recovery state hydrogels were immersed in distilled water for 2 days to remove residual monomers, and then dried in vacuum at 60 °C for 24 h. The dry hydrogels were immersed into medium at 37 °C and the swollen weight to ensure it won't absorb the culture medium during incubating with cells. Afterward, the hydrogel was printed into the cylinder with 1.0 mm height and 2.5 mm radius, which met the requirements from the ISO 10993-5 and ISO 10993-12 that the bottom area should be 10% of each well surface area of a 24-well plate for a direct test. For the cell incubation, 3T3 cells were cultured onto a 24-well plate (1 × 105 cells/ well) in complete DMEM (with high glucose and 10% fetal bovine serum supplemented) culture medium in a humidified atmosphere of 5% CO2 at 37 °C. Meanwhile, after cultivating the cells for 24 h, the medium was replaced and the hydrogel samples were added. The cells were incubated for another 12, 24 and 36 h, respectively, and the cell viability was determined by CCK-8 kits.
2.3. 3D printing The F127DA-alginate ink was loaded into the extrusion cylinder. The nozzle with an inner diameter of 0.4 mm was used for printing. A 3D model (designed by 3ds Max) was imported, sliced and internal structure was designed by the printer's matching software. The glass dish was placed on a printing platform at 60 °C, so that the material was pasted into the culture dish and did not collapse. Through several attempts, the nozzle speed of 15 mm/s and the pressure of 0.04 kPa were used to achieve the best coordination for printing. The temperature of the nozzle was set at 25 °C. A 3D bioprinting machine (Envisiontec, Germany) created the original shape of the hydrogels, and then they were exposed under UV light (Ex 365 nm, 0.06 mW/cm2, WFH-203, Shanghai Jingke Industrial Co. Ltd.) to fix their shape.
Cell viability (%) = [A (dosing) − A (blank) ]/[A (0
2.4. Shape memory performance
A(Dosing)
The shape memory performance of the F127DA-alginate hydrogels was evaluated at room temperature. The mesh of the square was folded and put into the 1% (w/v) CaCl2 solution for 5 min to fix the temporary shape, and then transferred to the 2% (w/v) Na2CO3 solution to remove Ca2 + to restore its shape. The temporary state sample was immersed in distilled water to examine the stability of the temporary shape.
A(blank)
dosing)
− A (blank) ] ∗100%
Absorbance of wells with cells, CCK-8 solution and drug solution absorbance of wells with medium and CCK-8 solution without cells
2.9. In vitro degradation The in vitro degradation behavior of F127DA-alginate hydrogels was determined by weight loss measurements performed in the pH 7.4 phosphate buffer solution (PBS) at 37 °C [35]. Temporary and recovery states of the memory hydrogels were the test objects in in vitro degradation. Then they were placed in 50 mL PBS buffer, respectively. The quality of the hydrogel was weighed at each predetermined point in time. The studies were carried out in triplicate.
2.5. Rheological experiments of F127DA-alginate hydrogels Stress sweep and frequency sweep experiments were used to investigate the viscoelastic properties of F127DA-alginate hydrogels. Survey was performed by using a strain-controlled Rheumatics ARES rheometer fitted with a plate-plate tool whose diameter is 20 mm with a gap of 1.0 mm. The storage modulus (G′) and loss modulus (G″) were measured as a function of frequency within a linear angle of viscoelasticity at 25 °C and the frequency from 0.1 to 100 Hz.
3. Results and discussion 3.1. F127DA-alginate ink fabrication
2.6. 3D printed of MTX-loaded F127DA-alginate ink
The 3D printer is an extruded type, so the viscosity of the printing ink affects the quality of the print. The shear viscosity of different concentrations of F127DA is shown in Fig. 2a. It shows that the viscosity value increased from 51.26 to 482.5 Pa s under the shear rate of 0.1 rad/s, and increased from 2.06 to 4.17 Pa s under the frequency of 100 rad/s. It conveys that the ink fabricated by the higher concentration of F127DA had a greater viscosity. F127DA is temperature sensitive [36], so it is advantageous to print shape and adsorb platform when the platform temperature is raised. In order to find the best print parameters, the ink was printed as lines at different platform temperatures. Fig.2b shows that 12% F127DA had a line expansion rate of 377.5% at 40 °C and 359.1% at 60 °C, and 14% F127DA was 280.0% and 267.5% at the same temperature. There is a 92 and 97 percentage point difference between the two groups, respectively. Line expansion rate difference between the two groups is significant because of the low concentration F127DA printing line is not sufficient to form a gel, so
The antitumor drug methotrexate (MTX) was loaded on the hydrogel. As MTX is hydrophobic, 1 mg of MTX was first dissolved in 0.1 mL of VP and then added by drop into 5 mL of print inks. The mixture was stirred for 4 h, and the whole process was kept in dark operation. The method of 3D printing was the same as that described in Section 2.3 of this paper. 2.7. In vitro drug release In order to evaluate the effect of the hydrogel internal structure, we compare the differences in the drug release between the 3D printing hydrogel (internal structure) and traditional manufacturing hydrogel (without internal structure). Both the drug-loaded hydrogels were in temporary state. In order to learn more about the release properties of 46
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 3. The shear modulus of original and recovery state F127DA-alginate hydrogels. (a) The shear modulus of original state F127DA-alginate hydrogels with different concentrations of alginate. (b) The shear modulus of recovery state F127DA-alginate hydrogels with different concentrations of alginate. Fig. 2. The value of shear viscosity and line expansion rates of inks with different concentrations of F127DA. (a) The shear viscosity of inks. (b) Print line expansion rates of different inks with gradient platform temperature.
ratio of 6% alginate was 73.03%, which was significantly lower than 2% and 4%. Then the storage modulus (G′) of the original state and recovery state was measured at 25 °C, respectively. Fig. 3a shows the shear modulus of the original state hydrogel with a different alginate. Fig. 3b shows the shear modulus of the recovery state hydrogel with a different alginate. We calculated the ratio of the shear modulus of recovery state to original state in the frequency range of 1 rad/s to 10 rad/s. The ratio of 2% alginate was 132.3% to 107.9%, 4% alginate was 112.4% to 97.8% and 6% alginate was 90.8% to 80.1%. The modulus ratio of 4% alginate hydrogel was closer to 100%. Compared with the other two groups the mechanical response rate was the highest. Through analysis of the experimental results, 4% alginate was the best concentration for shape memory hydrogel. We conclude that 16% (w/v) F127DA and 4% (w/v) alginate show good performance both in printing and shape memory behavior.
mobility is relatively large. At lower shear forces, the viscosity of 16% F127DA was lower than 18% F127DA and 20% F127DA, and the same pressure was used in printing. Therefore, the high-viscosity of F127DA (18% and 20%) extrusion speed will be slightly slower than 16% F127DA, so the 16% F127DA expansion rate will be slightly lower than these two groups. Through analysis of the experimental results, the expansion rate of 16% F127DA was found to be the lowest, so it was chosen for printing. Fig. 2b also shows that with the increase of the temperature of the platform, the line expansion rate gradually reduced, and the gap between the two groups became smaller and smaller. Group 60 °C and group 70 °C were relatively satisfactory; taking into account security, a lower temperature was used. To select the optimum alginate concentration, alginate 2%, 4% and 6% (w/v) ink containing an equal amount of F127DA were used to print and evaluate their shape memory cycles. Table 1 shows that the recovery ratios of the three groups are close to 100%, but the fixation
3.2. 3D printing and shape memory performance A hydrogel with an internal mesh structure was 3D printed and shown in Fig. 4. The internal mesh structure of the 3D model was designed by the printer's matching software. The glass dish was placed on a printing platform because the F127DA-alginate ink was pasted into the culture dish and did not collapse. The nozzle speed and the printed pressure achieved the best coordination for printing through a simple shape of the print test. Fig. 4a shows the network structure of the printed hydrogel; the gap between the grids was obvious, and the space diameter was about 2 mm. This proves the superiority of 3D printing,
Table 1 Value of fixity ratio and recovery ratio of different alginate. Hydrogel
Fixity ratio (%)
Recovery ratio (%)
F127DA (16%)-alginate (2%) F127DA (16%)-alginate (4%) F127DA (16%)-alginate (6%)
89.34 ± 3.34 81.47 ± 1.3 73.03 ± 5.63
97.22 ± 2.78 98.15 ± 2.62 99.07 ± 1.31
47
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 4. Photographs display the shape memory behavior of the F127DA-alginate hydrogels. (a) Printing shape. (b) UV 365 nm fixed original shape of hydrogels. (c) Temporary shape of the hydrogel was set in 1% CaCl2 solution and recovered in 2% Na2CO3 solution for 0 min.(d), (e) and (f) Hydrogels' shape recovered in 2% Na2CO3 solution. (g), (h) and (i) Temporary shape of hydrogels immersed in distilled water.
not only can it change the external form but also change the fine internal structure. Fig. 4b shows the 365 nm UV curing SMHs. Compared with the printed shape, the edge of the small gap has a slight collapse after 5 min of UV cross-linking. The reason was that a lower concentration of photosensitizer was used. Although increasing the concentration of photosensitizer can accelerate the cure rate of F127DA to get a better shape, it also introduces harmful ingredients. Another solution is to change the printing method to layer-by-layer curing. However, this print structure will affect the mechanical properties of hydrogels. Our aim is to produce a shape memory hydrogel with biocompatible and excellent mechanical properties, so we use as low a photosensitizer concentration as possible without affecting the hydrogel molding. And the mesh and shape of the hydrogels which was printed use our method was close to pre-designed. As expected, the F127DA-alginate hydrogels shape memory behavior was shown in Fig. 4. The sample was folded by external stress and then the sample was transferred rapidly into the 1% (w/v) CaCl2 solution for 5 min to fix the temporary shape. When the external stress was released, the sample mostly kept a temporary shape. Then the sample was transferred to the 2% (w/v) Na2CO3 solution to remove Ca2 + to restore its original shape. The result was shown in Fig. 4c. Fig.4d, 4e and 4f shows the shape recovery process for hydrogels. In the first 5 min (Fig. 4d), the diagonal opened and the shape began to gradually recover. Fig. 4e was restored at 10 min, until the first 30 min (Fig. 4f) that the shape was almost completely reduced. Although the F127DA-alginate hydrogel takes 30 min to restore the original form, in the first 10 min the reduction rate was over 90%. It proved that F127DA-
alginate hydrogel had strong shape memory performance. In order to further explore the shape memory behavior of F127DAalginate hydrogels, the second shape memory cycle was followed. The results are not shown because the fixed ratio was low. During the process of hydrogel recovery, as the alginate network structure was opened, a portion of the free alginate was dissipated in the solution. When the fixation was performed again, the F127DA network remained stable so that the crosslinking strength of the Ca2 +-alginate was insufficient to support the temporary shape of the hydrogel. In order to prove the temporary state of the stability of the hydrogel, the temporary state sample was immersed in distilled water. The result was shown in Fig. 4h and Fig. 4i. There was negligible change of the temporary shape, and the hydrogels did not swell after 48 h. Because of the high density of dual network structure, the swelling tendency was suppressed. 3.3. Rheological experiments of F127DA-alginate hydrogels There were four kinds of state of F127DA-alginate hydrogels in the whole shape memory cycle process which was printing state, original state, temporary state and recovery state, respectively. The elastic modulus (G′) and loss modulus (G″) of these four states were measured to further explore the hydrogel mechanical properties of the shape memory cycle process. The frequency dependence of the G′ and G″ of the F127DA-alginate hydrogels at 25 °C were shown in Fig. 5. The values of elastic and loss modulus of printing state were shown in Fig. 5a, where the value of G″ was greater than G′. This indicates that the printing state was a viscous body that does not form a hydrogel. 48
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 5. The elastic modulus (G′) and loss modulus (G″) of four states in shape memory cycle. (a) The G′ and G″ of printing state hydrogels. (b) The G′ and G″ of original state hydrogels. (c) The G′ and G″ of temporary state hydrogels. (d) The G′ and G″ of recovery state hydrogels.
3.4. Drug loading and in vitro release
Under the irradiation of 365 nm wavelengths, the photoinitiator excited F127DA crosslinked a stable network which fixed the original shape of the hydrogel. The value of elastic and loss modulus of the original state was shown in Fig. 5b. It shows that the elastic modulus value increased from 17.7 to 22.7 kPa under the frequency range from 1 to 10 rad/s. The loss modulus value ranged from 3.4 to 3.9 kPa within the same frequency ranges. The value of G′ was greater than G″ over the entire frequency range, which is consistent with the solid elastic properties of the hydrogel [37]. Fig. 5c shows the temporary state of memory hydrogels after Ca2 + cross-linking. The crosslinking network of sodium alginate provides support for the fixation of the temporary shape. The elastic modulus value increased from 35.0 to 42.0 kPa under the frequency range from 1 to 10 rad/s. The values of G′ was significantly increased compared to the original state. Such changing tendency of the elastic modulus is attributed to the ion-crosslink which provided the secondary network within the hydrogels. The Ca2 +-alginate and F127DA network form a dual network structure. Fig. 5d shows that the gel in the Na2CO3 solution returns to its original state. The Na2CO3 solution destroyed the cross-linked network structure of alginate and Ca2 +. At this time, there was only the F127DA network structure in the hydrogel, and the hydrogel recovered its original state under the action of Ca2 +. The elastic modulus value increased from 19.9 to 22.2 k Pa under the frequency range from 1 to 10 rad/s. The average ratio of the shear modulus of the recovery state to original state was 104.34%. The G′ value of the recovery state was greater than the original state. This was because there was still a small amount of alginate and Ca2 + crosslinked structures inside the hydrogels. And these structures did not affect the recovery state of hydrogels. It indicates that our hydrogel mechanical properties remain stable after the shape memory cycle. Unlike other shape memory hydrogels, our hydrogels display excellent characteristics because the shape and mechanical properties can be restored to the original state. Besides, the formation of dual network structure can not only improve the mechanical properties, but also contribute to stabilizing the temporary shape of the hydrogels.
The application of SMHs as a drug delivery device is achieved through a load of MTX, a clinically commonly used anticancer drug. The drug molecules were encapsulated in the hydrogel by the formation of the dual network structure. As MTX is hydrophobic, MTX was first dissolved in VP that it could be better distributed in the printing ink. The drug loading rate of the F127DA-agilent hydrogel was 41.67 μg/ mg. In order to evaluate the effect of the hydrogel internal structure, a drug loaded hydrogel with internal mesh structure was printed. Then, the weight of the same drug loaded hydrogel (without internal structure) was made by traditional manufacturing methods. The drug loaded hydrogels were placed into PBS for the release of the experiments in vitro while the result is shown in Fig. 6. The cumulative dose of drug release in the 3D printing group was about 80% within 6 h, while the traditional manufacturing group was about 50% at 6 h. The drug release properties were similar about both groups but the samples made by 3D-printing were able to release more MTX in the same period comparing to the other group. The design of the internal mesh structure increases the surface area ratio of the drug loaded hydrogel which increases the rate of drug release. SMPs may distribute to a more stable and high quantity release behavior. The performance of rapid drug release provides potential applications for local release anesthesia or hemostasis drug in clinical operation. Therefore, it is interesting to note that 3D printing could provide a better structure for us to control the molecular releasing behavior. Fig. 6 also showed that cumulative release of the temporary state was about 80% and the recovery state was about 40%. The release of the temporary state was about 40% higher than the recovery state. It was due to the shape recovery process while the alginate-Ca2 + network opened and the MTX was released. The recovery process was about 30 min. This rapid release under certain conditions provided a new possibility for the controlled release of the drug. 49
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
3.5. Cytotoxicity The shape memory hydrogel remains in the temporary state when it was used. It recovers to its original shape (recovery state) when its application ends. So the temporary and recovery states of F127DA-alginate shape memory hydrogel were the subjects of cytotoxicity assays. The in vitro cytotoxicity of the F127DA-alginate hydrogels was evaluated on 3T3 cells by CCK-8 assays. Fig. 7 provided the evidence of the cell viability of 3T3 cells. Regardless temporary or recovery state cell viability was more than 95% at 24 and 36 h. Alginate as a class of polysaccharides can provide certain nutrients to cells to promote cell growth. Low concentrations of photosensitizer concentrations demonstrate less damage to cells. The in vitro cytotoxicity of hydrogels demonstrates its excellent biological properties, increasing the potential of memory hydrogels in biomedical applications. 3.6. In vitro degradation Degradability of material determines the method of its application. The in vitro degradation behaviors of temporary and recovery states of the memory hydrogels were determined by weight loss measurements. As shown in Fig. 8, the sample quality in the first four days showed a downward trend either for the temporary or recovery state. This part of the mass loss was the alginate in the hydrogel because it degrades in PBS. Fig. 8 also shows that the degradation rate of the recovery state was faster than the temporary state. The temporary state was more stable than the recovery state because of the Ca2 + cross-linked alginate. In the next few days, the quality of the hydrogel remained stable and without degradation. This is because the F127DA photo-crosslinking has formed a stable network structure which was not degraded in PBS. Both the weights of temporary and recovery states remained above 85% after 7 days of degradation in PBS. It shows that the hydrogel does not degrade in vitro and can maintain its original morphology. Excellent biocompatibility and ability to maintain the original shape provide the possibility for the hydrogel in the body for short-term implantation.
Fig. 6. In vitro MTX release profiles from the hydrogels.
4. Conclusions In summary, we show the shape memory hydrogels produced by 3D printing. The 3D printing method not only adjusts the external shape of the gel, but also adjusts its internal structure. The internal structure of the hydrogel has the function of rapid drug release. The F127DA UV cross-linking network mechanism for the gel structure provides mechanical support, while providing the basis for 3D printing. AlginateCa2 + coordination is the key to the shape memory function of the hydrogels. SMHs have a high recovery ratio in a short time, and their mechanical properties can also be basically restored. This excellent biocompatibility shape memory hydrogel, which can change its internal structure, may have applications in actuators, tissue engineering or drug delivery in clinical surgery.
Fig. 7. The cell viability results of F127DA-alginate hydrogels.
5. Acknowledgments This work was financially supported by the fund from the Guangzhou Science and Technology Program (201607010127), the Natural Science Foundation of Guangdong Province (2014A030313361), the National Natural Science Foundation of China (51573071), the Pearl River S&T Nova Program of Guangzhou (201506010069) and the Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering. Fig. 8. In vitro degradation results of F127DA-alginate hydrogels.
References [1] Y. Si, L. Wang, X. Wang, et al., Ultrahigh-water-content, superelastic, and shapememory nanofiber-assembled hydrogels exhibiting pressure-responsive conductivity, Adv. Mater. 29 (24) (2017) 1700339.
50
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
regeneration pathways, Adv. Funct. Mater. 25 (39) (2015) 6205. [20] W.D.A. Sun, B. Starly, J. Nam, Computer-aided tissue engineering: overview, scope and challenges [review], Biotechnol. Appl. Biochem. 39 (1) (2004) 29–47. [21] S.M. Peltola, F.P. Melchels, D.W. Grijpma, et al., A review of rapid prototyping techniques for tissue engineering purposes, Ann. Med. 40 (4) (2008) 268–280. [22] M. Zarek, M. Layani, I. Cooperstein, et al., 3D printing of shape memory polymers for flexible electronic devices, Adv. Mater. 28 (22) (2016) (p. 4449–+). [23] J.P.K. Armstrong, M. Burke, B.M. Carter, et al., 3D bioprinting using a templated porous bioink, Adv. Healthc. Mater. 5 (14) (2016) 1724–1730. [24] M.K. Gupta, F. Meng, B.N. Johnson, et al., 3D printed programmable release capsules, Nano Lett. 15 (8) (2015) 5321. [25] H.J. Chung, Y. Lee, T.G. Park, Thermo-sensitive and biodegradable hydrogels based on stereocomplexed pluronic multi-block copolymers for controlled protein delivery, J. Control. Release 127 (1) (2008) 22–30. [26] Y. Sun, S. Liu, G. Du, et al., Multi-responsive and tough hydrogels based on triblock copolymer micelles as multi-functional macro-crosslinkers, Chem. Commun. 51 (40) (2015) 8512–8515. [27] Y.F. Liu, W.F. Zhou, Q. Zhou, et al., F127DA micelle cross-linked PAACA hydrogels with highly stretchable, puncture resistant and self-healing properties, RSC Adv. 7 (47) (2017) 29489–29495. [28] S. Hong, D. Sycks, H.F. Chan, et al., 3D printing of highly stretchable and tough hydrogels into complex, cellularized structures, Adv. Mater. 27 (27) (2015) 4035. [29] S.T. Bendtsen, S.P. Quinnell, W. Mei, Development of a novel alginate-polyvinyl alcohol-hydroxyapatite hydrogel for 3D bioprinting bone tissue engineered scaffolds, J. Biomed. Mater. Res. A 105 (5) (2017) 1457. [30] Y.X. Luo, G.L. Luo, M. Gelinsky, et al., 3D bioprinting scaffold using alginate/ polyvinyl alcohol bioinks, Mater. Lett. 189 (2017) 295–298. [31] X.X. Le, W. Lu, J. Zheng, et al., Stretchable supramolecular hydrogels with triple shape memory effect, Chem. Sci. 7 (11) (2016) 6715–6720. [32] W.J. Nan, W. Wang, H. Gao, et al., Fabrication of a shape memory hydrogel based on imidazole-zinc ion coordination for potential cell-encapsulating tubular scaffold application, Soft Matter 9 (1) (2013) 132–137. [33] Y.N. Chen, L. Peng, T. Liu, et al., Poly(vinyl alcohol)–tannic acid hydrogels with excellent mechanical properties and shape memory behaviors, ACS Appl. Mater. Interfaces 8 (40) (2016) 27199. [34] D. Ma, L.M. Zhang, C. Yang, et al., UV photopolymerized hydrogels with beta-cyclodextrin moieties, J. Polym. Res. 15 (4) (2008) 301–307. [35] X.J. Han, Z.Q. Dong, M.M. Fan, et al., pH-induced shape-memory polymers, Macromol. Rapid Commun. 33 (12) (2012) 1055. [36] H. Meng, P. Xiao, J. Gu, et al., Self-healable macro-/microscopic shape memory hydrogels based on supramolecular interactions, Chem. Commun. 50 (82) (2014) 12277. [37] A. Yasin, H. Li, Z. Lu, et al., A shape memory hydrogel induced by the interactions between metal ions and phosphate, Soft Matter 10 (7) (2014) 972.
[2] D. Ratna, J. Karger-Kocsis, Recent advances in shape memory polymers and composites: a review, J. Mater. Sci. 43 (1) (2008) 254–269. [3] K. Inomata, T. Terahama, R. Sekoguchi, et al., Shape memory properties of polypeptide hydrogels having hydrophobic alkyl side chains, Polymer 53 (15) (2012) 3281–3286. [4] A. Lendlein, R. Langer, Biodegradable, elastic shape-memory polymers for potential biomedical applications, Science 296 (5573) (2002) 1673. [5] C.M. Yakacki, R. Shandas, D. Safranski, et al., Strong, tailored, biocompatible shape-memory polymer networks, Adv. Funct. Mater. 18 (16) (2008) 2428–2435. [6] R.Z. Alavijeh, P. Shokrollahi, J. Barzin, A thermally and water activated shape memory gelatin physical hydrogel, with a gel point above the physiological temperature, for biomedical applications, J. Mater. Chem. B 5 (12) (2017) 2302–2314. [7] Y. Hu, W. Guo, J.S. Kahn, et al., A shape-memory DNA-based hydrogel exhibiting two internal memories, Angew. Chem. Int. Ed. 55 (13) (2016) 4210. [8] Y.W. Hu, C.H. Lu, W.W. Guo, et al., A shape memory acrylamide/DNA hydrogel exhibiting switchable dual pH-responsiveness, Adv. Funct. Mater. 25 (44) (2015) 6867–6874. [9] C.M. Yakacki, R. Shandas, C. Lanning, et al., Unconstrained recovery characterization of shape-memory polymer networks for cardiovascular applications, Biomaterials 28 (14) (2007) 2255–2263. [10] C. Wischke, A.T. Neffe, S. Steuer, et al., Evaluation of a degradable shape-memory polymer network as matrix for controlled drug release, J. Control. Release 138 (3) (2009) 243–250. [11] A. Goyanes, P.R. Martinez, A. Buanz, et al., Effect of geometry on drug release from 3D printed tablets, Int. J. Pharm. 494 (2) (2015) 657–663. [12] A. Goyanes, M. Kobayashi, R. Martínez-Pacheco, et al., Fused-filament 3D printing of drug products: microstructure analysis and drug release characteristics of PVAbased caplets, Int. J. Pharm. 514 (1) (2016) 290–295. [13] N. Raja, H.S. Yun, A simultaneous 3D printing process for the fabrication of bioceramic and cell-laden hydrogel core/shell scaffolds with potential application in bone tissue regeneration, J. Mater. Chem. B 4 (27) (2016) 4707–4716. [14] I.G. Kim, J. Ko, H.R. Lee, et al., Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering, Biomaterials 85 (2016) 18–29. [15] A. Kumar, K.C. Nune, R.D. Misra, Biological functionality and mechanistic contribution of extracellular matrix-ornamented three dimensional Ti-6Al-4V mesh scaffolds, J. Biomed. Mater. Res. A 104 (11) (2016) 2751. [16] J.D. Erndt-Marino, S. Becerra-Bayona, R.E. Mcmahon, et al., Cell layer-electrospun mesh composites for coronary artery bypass grafts, J. Biomed. Mater. Res. A 104 (9) (2016) 2200. [17] Q. Lin, X. Hou, C. Ke, Ring shuttling controls macroscopic motion in a three-dimensional printed polyrotaxane monolith, Angew. Chem. Int. Ed. 56 (16) (2017). [18] A. Ambrosi, M. Pumera, 3D-printing technologies for electrochemical applications, Chem. Soc. Rev. 45 (10) (2016) 2740. [19] B.N. Johnson, K.Z. Lancaster, G. Zhen, et al., 3D printed anatomical nerve
51
Contents lists available at ScienceDirect
Materials Science & Engineering C journal homepage: www.elsevier.com/locate/msec
Three-dimensional printing of shape memory hydrogels with internal structure for drug delivery
T
Yongzhou Wanga, Ying Miaoa, Jieling Zhanga, Jian Ping Wub, Thomas Brett Kirkb, Jiake Xuc, ⁎ ⁎ Dong Maa, , Wei Xuea, a
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China 3D Imaging and Bioengineering Laboratory, Department of Mechanical Engineering, Curtin University, Perth, Australia c The School of Pathology and Laboratory Medicine, University of Western Australia, Perth, Australia b
A R T I C L E I N F O
A B S T R A C T
Keywords: 3D printing Shape memory hydrogels Internal structure Rapid drug release
Hydrogels with shape memory behavior and internal structure have wide applications in fields ranging from tissue engineering and medical instruments to drug delivery; however, creating the hydrogels has proven to be extremely challenging. This study presents a three-dimensional (3D) printing technology to fabricate the shape memory hydrogels with internal structure (SMHs) by combining sodium alginate (alginate) and pluronic F127 diacrylate macromer (F127DA). SMHs were constituted by a dual network structure. One is a stable network which is formed by F127DA photo-crosslinking; the other one is a reversible network which is formed by Ca2 + cross-linked alginate. SMHs recovery ratio was 98.15% in 10 min after Ca2 + was removed in the Na2CO3 solution, and the elastic modulus remains essentially stable after the shape memory cycle. It showed that the drug releasing rate is more rapid compared with traditional drug-loaded hydrogels in in vitro experiments. The viability of 3T3 fibroblasts remained intact which revealed its excellent biocompatibility. Therefore, SMHs have a huge prospect for application in drug carriers and tissue engineering scaffold.
1. Introduction Shape memory hydrogels are a class of gel that can restore its original form in the presence of external stimuli [1–3]. Lendlein reported a PCL-based shape memory polymer (SMPs) and demonstrated its potential in medical applications [4]. Since then more and more research focused on the development of SMPs biocompatible applications [5,6]. Willner et al. have developed DNA hydrogels with shape memory performance by regulating pH value [7,8]. Yakacki et al. have proposed a shape memory polymer for cardiovascular stent [9]. Shape-memory in drug controlled release devices was reported by Wischke et al. [10]. Drug release from the tablets depended on the surface area to volume ratio [11]. The internal structure of the hydrogel can change its surface area volume ratio to have a positive effect on drug controlled release [12]. The internal structure of the hydrogel is widely used in cell culture [13] and tissue engineering scaffolds [14,15] because it uses less material to provide space support for cell growth. For example, ErndtMarino et al. reported a cell layer-electrospun mesh internal structure as coronary artery bypass grafts [16]. Lin et al. reported 3D printing polyrotaxane-based lattice cubes which are capable of converting the chemical energy input into mechanical work; it is also proved that the
⁎
polymer without the internal structure cannot achieve this conversion effect [17]. So the shape memory hydrogel with internal structure has a huge potential value in the medical field. But there is still no effective way to control the internal structure of the shape memory hydrogels. To address this issue, an attractive strategy would be the introduction of a 3D printing technique applied to the manufacturing process of shape memory hydrogels. 3D printing is recognized as a promising technology since it was developed by Charles Hull in the early 1980s [18]. Nowadays, the development of computer-aided design and image processing enable 3D printing to reconstruct an accurate physical model [19–21]. 3D printing technology used in the manufacture of SMPs is also gradually reported [17]. A previous work carried out by Zarek et al. described 3D printing of shape memory polymers that can be used in flexible electronic devices [22]. Integrating 3D printability to SMPs will allow us to explore the possibility of 3D printing SMHs. Here, we report the design and synthesis of printable hydrogels (Fig. 1) which are composed of alginate and pluronic F127 (EO100PO65-EO100; PEO = poly (ethylene oxide), PPO = poly (propylene oxide)). F127 is a common material in 3D printing areas and it has a sol–gel transition near physiological temperatures [23,24]. Pluronic F127 diacrylate macromer (F127DA) also has this characteristic
Corresponding authors. E-mail addresses: [email protected] (D. Ma), [email protected] (W. Xue).
https://doi.org/10.1016/j.msec.2017.11.025 Received 31 July 2017; Received in revised form 11 October 2017; Accepted 22 November 2017 Available online 23 November 2017 0928-4931/ © 2017 Elsevier B.V. All rights reserved.
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 1. The 3D printed shape memory hydrogel illustration. (a), (b), (c) The molecular mechanism of 3D printed shape memory hydrogels. (d) 3D printing memory hydrogel macro performance.
used as received without further purification.
because it retains a PEO-PPO-PEO tri-block copolymer structure [25]. F127DA has been reported for the manufacture of multi-responsive [26] and self-healing [27] hydrogels with excellent mechanical properties. F127DA can be crosslinked under the excitation of a photoinitiator to form a stable hydrogel at room temperature. The hydrogel was printed (printing state) by a 3D printer. The original shape of the hydrogel (original state) was formed by UV cross-linking of F127DA. Alginate as a natural polysaccharide which can form a high viscosity aqueous solution was widely used in 3D printing because of its good biocompatibility [28–30]. We selected alginate as the other material because the chelating of alginate with Ca2 + imparts a shape memory function to hydrogels [31]. Ca2 + cross-linked sodium alginate fixes the temporary shape of the hydrogel (temporary state). The hydrogel was restored to its original shape (recovery state) by Ca2 + substitution which was by destroyed the cross-linked network of calcium alginate. This study explores printing ink and printing parameters to create the excellent shape memory behavior of SMHs. The printing ink was designed by combining F127DA and alginate. The printing parameters were explored by calculating the expansion rate of the print lines. The shear modulus of hydrogels in the shape memory behavior cycle was analyzed by rheological experiments. It is also reported here that SMPs have good biocompatibility and can be rapidly released as a drug carrier.
2.2. F127DA-alginate ink fabrication A critical first step was to develop printing ink, while the F127DA component was the principle determinant of initial print quality. Therefore, it was necessary to first determine the content of component F127DA. The ink in 3D printing compatibility experiment was tested over a range of 10%, 12%, 14%, 16%, 18% and 20% (w/v) F127DA with 4% (w/v) alginate. The shear viscosity of different concentrations of F127DA was measured at 25 °C. Then the ink was printed into lines to determine its expansion rate at different platform temperatures. The line expansion rate is the ratio of the diameter of the print line to the nozzle diameter. The line diameter was measured three times by a micrometer. Alginate-Ca2 + coordination was the key to the shape memory function of the hydrogels. Accordingly, alginate was 2%, 4% and 6% (w/v) in the ink containing an equal amount F127DA, and the ink was used to print and evaluated its shape memory cycles to select the optimum alginate concentration. The quantitative shape memory cycle was determined according to the reported method [32]. Briefly, straight shapes were printed and cured with UV light for 3 min to form a hydrogel. It was bent into a U-form and immersed into 1% (w/v) CaCl2 solution for 2 min. Then the deformed hydrogel was transferred into 2% (w/v) Na2CO3 solution to remove Ca2 + in the hydrogel. The shape memory cycles were evaluated by measuring the angle at specific time points. The shape fixity ratio (Rf) and shape recovery ratio (Rr) were defined by the following equations [33]:
2. Experimental sections 2.1. Materials F127DA modified from F127, which includes material, pluronic F127 (Sigma, USA), acrylo acryloyl chloride (Aladdin, USA), triethylamine and dichloromethane (Tianjin Damao Chemical Reagents Company, China). 2,2-Dimethoxy-2-phenylacetophenone (DMPA) (Acros, USA) was dissolved in 1-vinyl-2-pyrrolidone (VP) (Aladdin, USA) as a photoinitiator. Alginate and methotrexate (MTX) were both purchased from Aladdin. All other chemicals were analytical grade and
Rf = θt /θi ∗100% Rr = (θi − θf )/θi ∗100% While θi is the given angle, θt is the temporarily fixed angle and θf is the final angle. The contents of the two components in the ink were determined by 45
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
the hydrogels, the drug release of 3D printing hydrogel of recovery state was explored. The MTX-loading hydrogels were placed into a vial containing 10 mL PBS (pH = 7.4) at 37 °C. Then 2 mL release media was taken out and 2 mL fresh pre-warmed release media was added back at every predetermined time points. The amount of released MTX was analyzed by UV–Vis spectrophotometer. All release studies were carried out in triplicate.
the above experiment. F127DA was dissolved in deionized water at a low temperature environment to form a uniform solution, followed by adding sodium alginate by slow and even magnetic stirring, and finally adding the photosensitizer solution (20 μL/mL) stirring evenly. F127DA was modified from F127. It was synthesized according to the reported method [34]. Briefly, 3.2 g F127 (dried in vacuum at 40 °C for 12 h) was dissolved in 25 mL dry CH2Cl2 in a 250 mL round bottomed flask and cooled to 0 °C in an ice bath. Then, triethylamine and acryloyl chloride were added dropwise to the solution under continuous magnetic stirring for more than 30 min then stirring at 0 °C and at room temperature for 12 h, respectively. After filtering off the by-product triethylamine hydrochloride, the mixture was washed by an excess of dichloromethane. After drying at 40 °C under vacuum for 24 h, the F127DA was obtained with a yield of 89%. The photosensitizer solution was prepared by 0.1 g DMPA dissolved in 1 mL VP.
2.8. Cytotoxicity 3T3 cells were incubated with the hydrogel for testing the cytotoxicity in this work. For the samples' preparation, the temporary and recovery state hydrogels were immersed in distilled water for 2 days to remove residual monomers, and then dried in vacuum at 60 °C for 24 h. The dry hydrogels were immersed into medium at 37 °C and the swollen weight to ensure it won't absorb the culture medium during incubating with cells. Afterward, the hydrogel was printed into the cylinder with 1.0 mm height and 2.5 mm radius, which met the requirements from the ISO 10993-5 and ISO 10993-12 that the bottom area should be 10% of each well surface area of a 24-well plate for a direct test. For the cell incubation, 3T3 cells were cultured onto a 24-well plate (1 × 105 cells/ well) in complete DMEM (with high glucose and 10% fetal bovine serum supplemented) culture medium in a humidified atmosphere of 5% CO2 at 37 °C. Meanwhile, after cultivating the cells for 24 h, the medium was replaced and the hydrogel samples were added. The cells were incubated for another 12, 24 and 36 h, respectively, and the cell viability was determined by CCK-8 kits.
2.3. 3D printing The F127DA-alginate ink was loaded into the extrusion cylinder. The nozzle with an inner diameter of 0.4 mm was used for printing. A 3D model (designed by 3ds Max) was imported, sliced and internal structure was designed by the printer's matching software. The glass dish was placed on a printing platform at 60 °C, so that the material was pasted into the culture dish and did not collapse. Through several attempts, the nozzle speed of 15 mm/s and the pressure of 0.04 kPa were used to achieve the best coordination for printing. The temperature of the nozzle was set at 25 °C. A 3D bioprinting machine (Envisiontec, Germany) created the original shape of the hydrogels, and then they were exposed under UV light (Ex 365 nm, 0.06 mW/cm2, WFH-203, Shanghai Jingke Industrial Co. Ltd.) to fix their shape.
Cell viability (%) = [A (dosing) − A (blank) ]/[A (0
2.4. Shape memory performance
A(Dosing)
The shape memory performance of the F127DA-alginate hydrogels was evaluated at room temperature. The mesh of the square was folded and put into the 1% (w/v) CaCl2 solution for 5 min to fix the temporary shape, and then transferred to the 2% (w/v) Na2CO3 solution to remove Ca2 + to restore its shape. The temporary state sample was immersed in distilled water to examine the stability of the temporary shape.
A(blank)
dosing)
− A (blank) ] ∗100%
Absorbance of wells with cells, CCK-8 solution and drug solution absorbance of wells with medium and CCK-8 solution without cells
2.9. In vitro degradation The in vitro degradation behavior of F127DA-alginate hydrogels was determined by weight loss measurements performed in the pH 7.4 phosphate buffer solution (PBS) at 37 °C [35]. Temporary and recovery states of the memory hydrogels were the test objects in in vitro degradation. Then they were placed in 50 mL PBS buffer, respectively. The quality of the hydrogel was weighed at each predetermined point in time. The studies were carried out in triplicate.
2.5. Rheological experiments of F127DA-alginate hydrogels Stress sweep and frequency sweep experiments were used to investigate the viscoelastic properties of F127DA-alginate hydrogels. Survey was performed by using a strain-controlled Rheumatics ARES rheometer fitted with a plate-plate tool whose diameter is 20 mm with a gap of 1.0 mm. The storage modulus (G′) and loss modulus (G″) were measured as a function of frequency within a linear angle of viscoelasticity at 25 °C and the frequency from 0.1 to 100 Hz.
3. Results and discussion 3.1. F127DA-alginate ink fabrication
2.6. 3D printed of MTX-loaded F127DA-alginate ink
The 3D printer is an extruded type, so the viscosity of the printing ink affects the quality of the print. The shear viscosity of different concentrations of F127DA is shown in Fig. 2a. It shows that the viscosity value increased from 51.26 to 482.5 Pa s under the shear rate of 0.1 rad/s, and increased from 2.06 to 4.17 Pa s under the frequency of 100 rad/s. It conveys that the ink fabricated by the higher concentration of F127DA had a greater viscosity. F127DA is temperature sensitive [36], so it is advantageous to print shape and adsorb platform when the platform temperature is raised. In order to find the best print parameters, the ink was printed as lines at different platform temperatures. Fig.2b shows that 12% F127DA had a line expansion rate of 377.5% at 40 °C and 359.1% at 60 °C, and 14% F127DA was 280.0% and 267.5% at the same temperature. There is a 92 and 97 percentage point difference between the two groups, respectively. Line expansion rate difference between the two groups is significant because of the low concentration F127DA printing line is not sufficient to form a gel, so
The antitumor drug methotrexate (MTX) was loaded on the hydrogel. As MTX is hydrophobic, 1 mg of MTX was first dissolved in 0.1 mL of VP and then added by drop into 5 mL of print inks. The mixture was stirred for 4 h, and the whole process was kept in dark operation. The method of 3D printing was the same as that described in Section 2.3 of this paper. 2.7. In vitro drug release In order to evaluate the effect of the hydrogel internal structure, we compare the differences in the drug release between the 3D printing hydrogel (internal structure) and traditional manufacturing hydrogel (without internal structure). Both the drug-loaded hydrogels were in temporary state. In order to learn more about the release properties of 46
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 3. The shear modulus of original and recovery state F127DA-alginate hydrogels. (a) The shear modulus of original state F127DA-alginate hydrogels with different concentrations of alginate. (b) The shear modulus of recovery state F127DA-alginate hydrogels with different concentrations of alginate. Fig. 2. The value of shear viscosity and line expansion rates of inks with different concentrations of F127DA. (a) The shear viscosity of inks. (b) Print line expansion rates of different inks with gradient platform temperature.
ratio of 6% alginate was 73.03%, which was significantly lower than 2% and 4%. Then the storage modulus (G′) of the original state and recovery state was measured at 25 °C, respectively. Fig. 3a shows the shear modulus of the original state hydrogel with a different alginate. Fig. 3b shows the shear modulus of the recovery state hydrogel with a different alginate. We calculated the ratio of the shear modulus of recovery state to original state in the frequency range of 1 rad/s to 10 rad/s. The ratio of 2% alginate was 132.3% to 107.9%, 4% alginate was 112.4% to 97.8% and 6% alginate was 90.8% to 80.1%. The modulus ratio of 4% alginate hydrogel was closer to 100%. Compared with the other two groups the mechanical response rate was the highest. Through analysis of the experimental results, 4% alginate was the best concentration for shape memory hydrogel. We conclude that 16% (w/v) F127DA and 4% (w/v) alginate show good performance both in printing and shape memory behavior.
mobility is relatively large. At lower shear forces, the viscosity of 16% F127DA was lower than 18% F127DA and 20% F127DA, and the same pressure was used in printing. Therefore, the high-viscosity of F127DA (18% and 20%) extrusion speed will be slightly slower than 16% F127DA, so the 16% F127DA expansion rate will be slightly lower than these two groups. Through analysis of the experimental results, the expansion rate of 16% F127DA was found to be the lowest, so it was chosen for printing. Fig. 2b also shows that with the increase of the temperature of the platform, the line expansion rate gradually reduced, and the gap between the two groups became smaller and smaller. Group 60 °C and group 70 °C were relatively satisfactory; taking into account security, a lower temperature was used. To select the optimum alginate concentration, alginate 2%, 4% and 6% (w/v) ink containing an equal amount of F127DA were used to print and evaluate their shape memory cycles. Table 1 shows that the recovery ratios of the three groups are close to 100%, but the fixation
3.2. 3D printing and shape memory performance A hydrogel with an internal mesh structure was 3D printed and shown in Fig. 4. The internal mesh structure of the 3D model was designed by the printer's matching software. The glass dish was placed on a printing platform because the F127DA-alginate ink was pasted into the culture dish and did not collapse. The nozzle speed and the printed pressure achieved the best coordination for printing through a simple shape of the print test. Fig. 4a shows the network structure of the printed hydrogel; the gap between the grids was obvious, and the space diameter was about 2 mm. This proves the superiority of 3D printing,
Table 1 Value of fixity ratio and recovery ratio of different alginate. Hydrogel
Fixity ratio (%)
Recovery ratio (%)
F127DA (16%)-alginate (2%) F127DA (16%)-alginate (4%) F127DA (16%)-alginate (6%)
89.34 ± 3.34 81.47 ± 1.3 73.03 ± 5.63
97.22 ± 2.78 98.15 ± 2.62 99.07 ± 1.31
47
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 4. Photographs display the shape memory behavior of the F127DA-alginate hydrogels. (a) Printing shape. (b) UV 365 nm fixed original shape of hydrogels. (c) Temporary shape of the hydrogel was set in 1% CaCl2 solution and recovered in 2% Na2CO3 solution for 0 min.(d), (e) and (f) Hydrogels' shape recovered in 2% Na2CO3 solution. (g), (h) and (i) Temporary shape of hydrogels immersed in distilled water.
not only can it change the external form but also change the fine internal structure. Fig. 4b shows the 365 nm UV curing SMHs. Compared with the printed shape, the edge of the small gap has a slight collapse after 5 min of UV cross-linking. The reason was that a lower concentration of photosensitizer was used. Although increasing the concentration of photosensitizer can accelerate the cure rate of F127DA to get a better shape, it also introduces harmful ingredients. Another solution is to change the printing method to layer-by-layer curing. However, this print structure will affect the mechanical properties of hydrogels. Our aim is to produce a shape memory hydrogel with biocompatible and excellent mechanical properties, so we use as low a photosensitizer concentration as possible without affecting the hydrogel molding. And the mesh and shape of the hydrogels which was printed use our method was close to pre-designed. As expected, the F127DA-alginate hydrogels shape memory behavior was shown in Fig. 4. The sample was folded by external stress and then the sample was transferred rapidly into the 1% (w/v) CaCl2 solution for 5 min to fix the temporary shape. When the external stress was released, the sample mostly kept a temporary shape. Then the sample was transferred to the 2% (w/v) Na2CO3 solution to remove Ca2 + to restore its original shape. The result was shown in Fig. 4c. Fig.4d, 4e and 4f shows the shape recovery process for hydrogels. In the first 5 min (Fig. 4d), the diagonal opened and the shape began to gradually recover. Fig. 4e was restored at 10 min, until the first 30 min (Fig. 4f) that the shape was almost completely reduced. Although the F127DA-alginate hydrogel takes 30 min to restore the original form, in the first 10 min the reduction rate was over 90%. It proved that F127DA-
alginate hydrogel had strong shape memory performance. In order to further explore the shape memory behavior of F127DAalginate hydrogels, the second shape memory cycle was followed. The results are not shown because the fixed ratio was low. During the process of hydrogel recovery, as the alginate network structure was opened, a portion of the free alginate was dissipated in the solution. When the fixation was performed again, the F127DA network remained stable so that the crosslinking strength of the Ca2 +-alginate was insufficient to support the temporary shape of the hydrogel. In order to prove the temporary state of the stability of the hydrogel, the temporary state sample was immersed in distilled water. The result was shown in Fig. 4h and Fig. 4i. There was negligible change of the temporary shape, and the hydrogels did not swell after 48 h. Because of the high density of dual network structure, the swelling tendency was suppressed. 3.3. Rheological experiments of F127DA-alginate hydrogels There were four kinds of state of F127DA-alginate hydrogels in the whole shape memory cycle process which was printing state, original state, temporary state and recovery state, respectively. The elastic modulus (G′) and loss modulus (G″) of these four states were measured to further explore the hydrogel mechanical properties of the shape memory cycle process. The frequency dependence of the G′ and G″ of the F127DA-alginate hydrogels at 25 °C were shown in Fig. 5. The values of elastic and loss modulus of printing state were shown in Fig. 5a, where the value of G″ was greater than G′. This indicates that the printing state was a viscous body that does not form a hydrogel. 48
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
Fig. 5. The elastic modulus (G′) and loss modulus (G″) of four states in shape memory cycle. (a) The G′ and G″ of printing state hydrogels. (b) The G′ and G″ of original state hydrogels. (c) The G′ and G″ of temporary state hydrogels. (d) The G′ and G″ of recovery state hydrogels.
3.4. Drug loading and in vitro release
Under the irradiation of 365 nm wavelengths, the photoinitiator excited F127DA crosslinked a stable network which fixed the original shape of the hydrogel. The value of elastic and loss modulus of the original state was shown in Fig. 5b. It shows that the elastic modulus value increased from 17.7 to 22.7 kPa under the frequency range from 1 to 10 rad/s. The loss modulus value ranged from 3.4 to 3.9 kPa within the same frequency ranges. The value of G′ was greater than G″ over the entire frequency range, which is consistent with the solid elastic properties of the hydrogel [37]. Fig. 5c shows the temporary state of memory hydrogels after Ca2 + cross-linking. The crosslinking network of sodium alginate provides support for the fixation of the temporary shape. The elastic modulus value increased from 35.0 to 42.0 kPa under the frequency range from 1 to 10 rad/s. The values of G′ was significantly increased compared to the original state. Such changing tendency of the elastic modulus is attributed to the ion-crosslink which provided the secondary network within the hydrogels. The Ca2 +-alginate and F127DA network form a dual network structure. Fig. 5d shows that the gel in the Na2CO3 solution returns to its original state. The Na2CO3 solution destroyed the cross-linked network structure of alginate and Ca2 +. At this time, there was only the F127DA network structure in the hydrogel, and the hydrogel recovered its original state under the action of Ca2 +. The elastic modulus value increased from 19.9 to 22.2 k Pa under the frequency range from 1 to 10 rad/s. The average ratio of the shear modulus of the recovery state to original state was 104.34%. The G′ value of the recovery state was greater than the original state. This was because there was still a small amount of alginate and Ca2 + crosslinked structures inside the hydrogels. And these structures did not affect the recovery state of hydrogels. It indicates that our hydrogel mechanical properties remain stable after the shape memory cycle. Unlike other shape memory hydrogels, our hydrogels display excellent characteristics because the shape and mechanical properties can be restored to the original state. Besides, the formation of dual network structure can not only improve the mechanical properties, but also contribute to stabilizing the temporary shape of the hydrogels.
The application of SMHs as a drug delivery device is achieved through a load of MTX, a clinically commonly used anticancer drug. The drug molecules were encapsulated in the hydrogel by the formation of the dual network structure. As MTX is hydrophobic, MTX was first dissolved in VP that it could be better distributed in the printing ink. The drug loading rate of the F127DA-agilent hydrogel was 41.67 μg/ mg. In order to evaluate the effect of the hydrogel internal structure, a drug loaded hydrogel with internal mesh structure was printed. Then, the weight of the same drug loaded hydrogel (without internal structure) was made by traditional manufacturing methods. The drug loaded hydrogels were placed into PBS for the release of the experiments in vitro while the result is shown in Fig. 6. The cumulative dose of drug release in the 3D printing group was about 80% within 6 h, while the traditional manufacturing group was about 50% at 6 h. The drug release properties were similar about both groups but the samples made by 3D-printing were able to release more MTX in the same period comparing to the other group. The design of the internal mesh structure increases the surface area ratio of the drug loaded hydrogel which increases the rate of drug release. SMPs may distribute to a more stable and high quantity release behavior. The performance of rapid drug release provides potential applications for local release anesthesia or hemostasis drug in clinical operation. Therefore, it is interesting to note that 3D printing could provide a better structure for us to control the molecular releasing behavior. Fig. 6 also showed that cumulative release of the temporary state was about 80% and the recovery state was about 40%. The release of the temporary state was about 40% higher than the recovery state. It was due to the shape recovery process while the alginate-Ca2 + network opened and the MTX was released. The recovery process was about 30 min. This rapid release under certain conditions provided a new possibility for the controlled release of the drug. 49
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
3.5. Cytotoxicity The shape memory hydrogel remains in the temporary state when it was used. It recovers to its original shape (recovery state) when its application ends. So the temporary and recovery states of F127DA-alginate shape memory hydrogel were the subjects of cytotoxicity assays. The in vitro cytotoxicity of the F127DA-alginate hydrogels was evaluated on 3T3 cells by CCK-8 assays. Fig. 7 provided the evidence of the cell viability of 3T3 cells. Regardless temporary or recovery state cell viability was more than 95% at 24 and 36 h. Alginate as a class of polysaccharides can provide certain nutrients to cells to promote cell growth. Low concentrations of photosensitizer concentrations demonstrate less damage to cells. The in vitro cytotoxicity of hydrogels demonstrates its excellent biological properties, increasing the potential of memory hydrogels in biomedical applications. 3.6. In vitro degradation Degradability of material determines the method of its application. The in vitro degradation behaviors of temporary and recovery states of the memory hydrogels were determined by weight loss measurements. As shown in Fig. 8, the sample quality in the first four days showed a downward trend either for the temporary or recovery state. This part of the mass loss was the alginate in the hydrogel because it degrades in PBS. Fig. 8 also shows that the degradation rate of the recovery state was faster than the temporary state. The temporary state was more stable than the recovery state because of the Ca2 + cross-linked alginate. In the next few days, the quality of the hydrogel remained stable and without degradation. This is because the F127DA photo-crosslinking has formed a stable network structure which was not degraded in PBS. Both the weights of temporary and recovery states remained above 85% after 7 days of degradation in PBS. It shows that the hydrogel does not degrade in vitro and can maintain its original morphology. Excellent biocompatibility and ability to maintain the original shape provide the possibility for the hydrogel in the body for short-term implantation.
Fig. 6. In vitro MTX release profiles from the hydrogels.
4. Conclusions In summary, we show the shape memory hydrogels produced by 3D printing. The 3D printing method not only adjusts the external shape of the gel, but also adjusts its internal structure. The internal structure of the hydrogel has the function of rapid drug release. The F127DA UV cross-linking network mechanism for the gel structure provides mechanical support, while providing the basis for 3D printing. AlginateCa2 + coordination is the key to the shape memory function of the hydrogels. SMHs have a high recovery ratio in a short time, and their mechanical properties can also be basically restored. This excellent biocompatibility shape memory hydrogel, which can change its internal structure, may have applications in actuators, tissue engineering or drug delivery in clinical surgery.
Fig. 7. The cell viability results of F127DA-alginate hydrogels.
5. Acknowledgments This work was financially supported by the fund from the Guangzhou Science and Technology Program (201607010127), the Natural Science Foundation of Guangdong Province (2014A030313361), the National Natural Science Foundation of China (51573071), the Pearl River S&T Nova Program of Guangzhou (201506010069) and the Guangdong Provincial Key Laboratory of Construction and Detection in Tissue Engineering. Fig. 8. In vitro degradation results of F127DA-alginate hydrogels.
References [1] Y. Si, L. Wang, X. Wang, et al., Ultrahigh-water-content, superelastic, and shapememory nanofiber-assembled hydrogels exhibiting pressure-responsive conductivity, Adv. Mater. 29 (24) (2017) 1700339.
50
Materials Science & Engineering C 84 (2018) 44–51
Y. Wang et al.
regeneration pathways, Adv. Funct. Mater. 25 (39) (2015) 6205. [20] W.D.A. Sun, B. Starly, J. Nam, Computer-aided tissue engineering: overview, scope and challenges [review], Biotechnol. Appl. Biochem. 39 (1) (2004) 29–47. [21] S.M. Peltola, F.P. Melchels, D.W. Grijpma, et al., A review of rapid prototyping techniques for tissue engineering purposes, Ann. Med. 40 (4) (2008) 268–280. [22] M. Zarek, M. Layani, I. Cooperstein, et al., 3D printing of shape memory polymers for flexible electronic devices, Adv. Mater. 28 (22) (2016) (p. 4449–+). [23] J.P.K. Armstrong, M. Burke, B.M. Carter, et al., 3D bioprinting using a templated porous bioink, Adv. Healthc. Mater. 5 (14) (2016) 1724–1730. [24] M.K. Gupta, F. Meng, B.N. Johnson, et al., 3D printed programmable release capsules, Nano Lett. 15 (8) (2015) 5321. [25] H.J. Chung, Y. Lee, T.G. Park, Thermo-sensitive and biodegradable hydrogels based on stereocomplexed pluronic multi-block copolymers for controlled protein delivery, J. Control. Release 127 (1) (2008) 22–30. [26] Y. Sun, S. Liu, G. Du, et al., Multi-responsive and tough hydrogels based on triblock copolymer micelles as multi-functional macro-crosslinkers, Chem. Commun. 51 (40) (2015) 8512–8515. [27] Y.F. Liu, W.F. Zhou, Q. Zhou, et al., F127DA micelle cross-linked PAACA hydrogels with highly stretchable, puncture resistant and self-healing properties, RSC Adv. 7 (47) (2017) 29489–29495. [28] S. Hong, D. Sycks, H.F. Chan, et al., 3D printing of highly stretchable and tough hydrogels into complex, cellularized structures, Adv. Mater. 27 (27) (2015) 4035. [29] S.T. Bendtsen, S.P. Quinnell, W. Mei, Development of a novel alginate-polyvinyl alcohol-hydroxyapatite hydrogel for 3D bioprinting bone tissue engineered scaffolds, J. Biomed. Mater. Res. A 105 (5) (2017) 1457. [30] Y.X. Luo, G.L. Luo, M. Gelinsky, et al., 3D bioprinting scaffold using alginate/ polyvinyl alcohol bioinks, Mater. Lett. 189 (2017) 295–298. [31] X.X. Le, W. Lu, J. Zheng, et al., Stretchable supramolecular hydrogels with triple shape memory effect, Chem. Sci. 7 (11) (2016) 6715–6720. [32] W.J. Nan, W. Wang, H. Gao, et al., Fabrication of a shape memory hydrogel based on imidazole-zinc ion coordination for potential cell-encapsulating tubular scaffold application, Soft Matter 9 (1) (2013) 132–137. [33] Y.N. Chen, L. Peng, T. Liu, et al., Poly(vinyl alcohol)–tannic acid hydrogels with excellent mechanical properties and shape memory behaviors, ACS Appl. Mater. Interfaces 8 (40) (2016) 27199. [34] D. Ma, L.M. Zhang, C. Yang, et al., UV photopolymerized hydrogels with beta-cyclodextrin moieties, J. Polym. Res. 15 (4) (2008) 301–307. [35] X.J. Han, Z.Q. Dong, M.M. Fan, et al., pH-induced shape-memory polymers, Macromol. Rapid Commun. 33 (12) (2012) 1055. [36] H. Meng, P. Xiao, J. Gu, et al., Self-healable macro-/microscopic shape memory hydrogels based on supramolecular interactions, Chem. Commun. 50 (82) (2014) 12277. [37] A. Yasin, H. Li, Z. Lu, et al., A shape memory hydrogel induced by the interactions between metal ions and phosphate, Soft Matter 10 (7) (2014) 972.
[2] D. Ratna, J. Karger-Kocsis, Recent advances in shape memory polymers and composites: a review, J. Mater. Sci. 43 (1) (2008) 254–269. [3] K. Inomata, T. Terahama, R. Sekoguchi, et al., Shape memory properties of polypeptide hydrogels having hydrophobic alkyl side chains, Polymer 53 (15) (2012) 3281–3286. [4] A. Lendlein, R. Langer, Biodegradable, elastic shape-memory polymers for potential biomedical applications, Science 296 (5573) (2002) 1673. [5] C.M. Yakacki, R. Shandas, D. Safranski, et al., Strong, tailored, biocompatible shape-memory polymer networks, Adv. Funct. Mater. 18 (16) (2008) 2428–2435. [6] R.Z. Alavijeh, P. Shokrollahi, J. Barzin, A thermally and water activated shape memory gelatin physical hydrogel, with a gel point above the physiological temperature, for biomedical applications, J. Mater. Chem. B 5 (12) (2017) 2302–2314. [7] Y. Hu, W. Guo, J.S. Kahn, et al., A shape-memory DNA-based hydrogel exhibiting two internal memories, Angew. Chem. Int. Ed. 55 (13) (2016) 4210. [8] Y.W. Hu, C.H. Lu, W.W. Guo, et al., A shape memory acrylamide/DNA hydrogel exhibiting switchable dual pH-responsiveness, Adv. Funct. Mater. 25 (44) (2015) 6867–6874. [9] C.M. Yakacki, R. Shandas, C. Lanning, et al., Unconstrained recovery characterization of shape-memory polymer networks for cardiovascular applications, Biomaterials 28 (14) (2007) 2255–2263. [10] C. Wischke, A.T. Neffe, S. Steuer, et al., Evaluation of a degradable shape-memory polymer network as matrix for controlled drug release, J. Control. Release 138 (3) (2009) 243–250. [11] A. Goyanes, P.R. Martinez, A. Buanz, et al., Effect of geometry on drug release from 3D printed tablets, Int. J. Pharm. 494 (2) (2015) 657–663. [12] A. Goyanes, M. Kobayashi, R. Martínez-Pacheco, et al., Fused-filament 3D printing of drug products: microstructure analysis and drug release characteristics of PVAbased caplets, Int. J. Pharm. 514 (1) (2016) 290–295. [13] N. Raja, H.S. Yun, A simultaneous 3D printing process for the fabrication of bioceramic and cell-laden hydrogel core/shell scaffolds with potential application in bone tissue regeneration, J. Mater. Chem. B 4 (27) (2016) 4707–4716. [14] I.G. Kim, J. Ko, H.R. Lee, et al., Mesenchymal cells condensation-inducible mesh scaffolds for cartilage tissue engineering, Biomaterials 85 (2016) 18–29. [15] A. Kumar, K.C. Nune, R.D. Misra, Biological functionality and mechanistic contribution of extracellular matrix-ornamented three dimensional Ti-6Al-4V mesh scaffolds, J. Biomed. Mater. Res. A 104 (11) (2016) 2751. [16] J.D. Erndt-Marino, S. Becerra-Bayona, R.E. Mcmahon, et al., Cell layer-electrospun mesh composites for coronary artery bypass grafts, J. Biomed. Mater. Res. A 104 (9) (2016) 2200. [17] Q. Lin, X. Hou, C. Ke, Ring shuttling controls macroscopic motion in a three-dimensional printed polyrotaxane monolith, Angew. Chem. Int. Ed. 56 (16) (2017). [18] A. Ambrosi, M. Pumera, 3D-printing technologies for electrochemical applications, Chem. Soc. Rev. 45 (10) (2016) 2740. [19] B.N. Johnson, K.Z. Lancaster, G. Zhen, et al., 3D printed anatomical nerve
51