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Apr 5, 2019 - The materials used for repairing bone have been subdivided into several major categories: autografts, allografts, and xenografts.(1) All...
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Article Cite This: ACS Omega 2019, 4, 6382−6391

http://pubs.acs.org/journal/acsodf

Modification of 3‑D Porous Hydroxyapatite/Thermoplastic Polyurethane Composite Scaffolds for Reinforcing Interfacial Adhesion by Polydopamine Surface Coating Zifeng Zheng,†,‡ Zhixiang Cui,*,†,‡ Junhui Si,†,‡ Shengrui Yu,⊥ Qianting Wang,†,‡ Wenzhe Chen,†,‡ and Lih-Sheng Turng*,§,∥ †

School of Materials Science and Engineering, Fujian University of Technology, Fujian 350118, China Fujian Provincial Key Laboratory in Universities of Polymer Materials and Production, Fujian 350118, China § Wisconsin Institutes for Discovery, University of Wisconsin-Madison, Madison, Wisconsin 53715, United States ∥ Department of Mechanical Engineering, University of Wisconsin-Madison, Madison, Wisconsin 53706, United States ⊥ School of Mechanical and Electric Engineering, Jingdezhen Ceramic Institute, Jiangxi 333403, China

ACS Omega 2019.4:6382-6391. Downloaded from pubs.acs.org by 178.57.68.80 on 04/05/19. For personal use only.

‡

ABSTRACT: Composite tissue engineering scaffolds show good comprehensive properties, but improving their interfacial binding force is still a challenge. In this study, polydopamine (PDA) was used as a glue-like layer to solve the interface problem in three-dimensional (3-D) porous hydroxyapatite (HA)/thermoplastic polyurethane (TPU) composite scaffolds and to improve their comprehensive properties. The morphology, water absorption, porosity, hydrophilicity, mechanical properties, and cell viability of HA/TPU and PDA/HA/ TPU scaffolds were systematically studied. The scanning electron microscopy and X-ray photoelectron spectroscopy results showed that a uniform PDA coating layer successfully formed on the surface of 3-D porous HA/TPU composite scaffolds by dopamine self-polymerization. Moreover, the conjugation effect occurred between the PDA and HA/TPU matrix, resulting in an increase in the interfacial binding force of the 3-D PDA/HA/TPU scaffold. The hydrophilicity of the PDA/HA/TPU scaffolds was significantly higher than those of the TPU scaffolds. Both static and dynamic mechanical property tests revealed that the PDA/HA/TPU scaffold possessed excellent comprehensive mechanical properties, such as high strength, high stiffness, and good flexibility, due to the enhancement of the interfacial binding force. Compared with TPU and HA/TPU scaffolds, the addition of a PDA coating could effectively improve the attachment and viability of mouse embryonic osteoblasts cells (MC3T3-E1) incubated on PDA/HA/TPU scaffolds. These results indicate that the PDA/HA/TPU scaffolds possess great potential to be used as tissue engineering scaffolds, and that the PDA coating could be a simple, practical, effective, and universal approach to modify composite scaffolds.

1. INTRODUCTION Bone defect repairs have been the ultimate goal of orthopedic surgery from ancient times to the present. The materials used for repairing bone have been subdivided into several major categories: autografts, allografts, and xenografts.1 Allografts and xenografts often suffer from the risk of disease transmission and immunoreactions.2 Autografts have osteoinductive, osteoconductive, and osteogenic characteristics but carry the limitations of morbidity at the harvesting site and limited availability.3 Given these problems, tissue engineering is a potential method to decrease the limitations of traditional grafts and enhance the repair process of bone defects and fractures. Bone tissue engineering scaffolds with three-dimensional (3-D) porous structures would serve as templates for culturing cells and promoting the reconstruction of bone tissue.4 Therefore, artificial bone substitutes, which serve the composition, bulk mechanical, and biological requirements of target tissues, have gained much attention. The fabrication of highly biomimetic 3-D bone tissue engineering scaffolds, which can mimic the natural bone matrix in composition, micro© 2019 American Chemical Society

structure, and mechanical strength for load-bearing applications, are still a challenge.5 Bioactive materials have been widely explored for the development of scaffold designs and processing, concentrating on the multitudinous needs in bone regeneration in the past decades. More and more attention has been given to bioceramics due to their ability to form a bone-like apatite phase that can strongly bond with bone or soft tissues.6−8 In particular, many reports have shown that hydroxyapatite (HA) has excellent osteoconductive and osteoinductive properties.9,10 However, for bioceramics, the disadvantages of high brittleness and poor formability have restricted its application.11 To overcome these limitations, a polymer with good elastic properties was used to prepare the HA/polymer composite scaffold. To date, various HA/polymer composite scaffolds, such as HA/silk, HA/hyaluronic acid, HA/poly(LReceived: February 18, 2019 Accepted: March 28, 2019 Published: April 5, 2019 6382

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lactic acid), HA/poly(lactic-co-glycolic acid), HA/polycaprolactone, HA/chitosan, HA/collagen, and so on, have been fabricated and have exhibited excellent potential for clinical applications.12−14 However, for most of the polymer/HA composite scaffolds, almost all of the HA nanoparticles were trapped in the polymer matrix, resulting in a decrease in the interaction between the cells and the bioactive HA nanoparticles. To overcome these problems, one of the main purpose of this study was to fabricate a 3-D porous thermoplastic polyurethane (TPU) scaffold modified with HA nanoparticles that resided on the surface instead of being trapped inside. TPU and its copolymers have been extensively used for fabricating tissue engineering scaffolds due to their advantages of high abrasion and chemical resistance, excellent elastic properties, adjustable mechanical properties, and good biodegradation rates.15 Nevertheless, the performance of TPU scaffolds has been limited by the lack of cell recognition sites, poor hydrophilicity, and low osteoconductive and osteoinductive properties. To overcome the disadvantages of TPU scaffolds, HA nanoparticles were added to improve the biochemical properties. In our laboratory’s previous research,16 the TPU/HA scaffold was fabricated using the thermally induced phase-separation method. The results showed that the mechanical properties and biocompatibility of the TPU/HA scaffolds were significantly higher than those of the TPU scaffolds due to the addition of HA particles. However, the interfacial binding force between the TPU matrix and the HA particles was not strong enough due to the poor material compatibility between the polymer and the ceramic,17 which caused the HA particles to fall off the TPU matrix after a period of time in biological fluids. In recent years, polydopamine (PDA) has attracted more and more attention due to its unique adhesion ability, which has a similar chemical structure to the adhesive proteins secreted by mussels. PDA can strongly adhere to all types of materials, such as oxides, polymers, metals, ceramics, and semiconductors, because of its rich catechol and amine functional groups. Therefore, to further improve the interfacial adhesion between the TPU matrix and the HA particles, one of the main objectives of this study was to introduce a PDA coating as a glue-like adhesive layer. Moreover, PDA has excellent biocompatibility because it is a neurotransmitter, a material that transmits messages from one cell to another. Herein, we developed a direct approach based on PDA deposition to immobilize HA nanoparticles on a 3-D TPU porous scaffold. The PDA coating not only increased the interfacial binding force between the HA particles and the TPU matrix, it also increased the biocompatibility of the HA/ TPU scaffold. The morphology, water absorption, porosity, hydrophilicity, mechanical properties, and cell viability of the HA/TPU and the PDA/HA/TPU scaffolds were systematically researched.

Figure 1. Morphology of TPU (a, b), HA/TPU (c, d), and PDA/ HA/TPU (e, f) scaffolds. The inset digital pictures are the surface (a1, c1, e1) and cross section (b1, d1, f1) for TPU (a1, b1), HA/TPU (c1, d1), and PDA/HA/TPU (e1, f1) scaffolds. The scale bar for (a, c, e) and (b, d, f) are 500 and 2.5 μm, respectively.

can be concluded that the surface modification process of ultrasonication and coating had no significant effect on the pore size and structure. From Figure 1b,d,f, the magnification images showed that the surface of the TPU scaffolds was smooth. Meanwhile, there were a lot of HA particles homogeneously immobilized on the surface of the HA/TPU scaffold, thereby increasing its surface roughness. Moreover, there were many additional PDA particles, which are larger than HA particles, on the surface of the PDA/HA/TPU scaffold in comparison with that of the HA/TPU scaffold, resulting in the smaller HA particles to disappear on the surface, which means the HA particles were completely covered by the PDA particles. The surface roughness of the HA/TPU and PDA/HA/TPU scaffolds was larger in comparison with that of the TPU scaffold, which means that the HA/TPU and PDA/HA/TPU scaffolds would have good biocompatibility because the largest surface roughness had better ability in terms of cell attachment and cell proliferation.19 After the PDA coating, both the surface and the interior of the PDA/HA/TPU scaffold turned from white to black, as the digital pictures show in Figure 1e1,f1. This result further confirmed that the PDA particles were successfully formed on the surface of the 3-D porous scaffolds by dopamine self-polymerization. The introduced PDA coating acted as a glue-like adhesive layer to adhere the HA particles to the TPU matrix and to improve the interfacial adhesion of the 3-D porous scaffolds. Furthermore, the porosity of the TPU, HA/ TPU, and PDA/HA/TPU scaffolds was calculated and the results were about 91.3, 87.1, and 80.7%, respectively. Although the porosity of the PDA/HA/TPU scaffold decreased compared with that of the TPU scaffold, it was still more than 80%. 2.2. Surface Chemical Analysis of Scaffolds. The typical X-ray photoelectron spectroscopy (XPS) spectra of neat TPU, HA/TPU, and PDA/HA/TPU scaffolds for C 1s, N 1s, O 1s, Ca 2p, and P 2p are shown in Figure 2. From Figure 2a−c, it can be seen that, for all TPU, HA/TPU, and PDA/ HA/TPU scaffolds, three peaks at 532.0, 400.0, and 285.0 eV

2. RESULTS AND DISCUSSION 2.1. Morphology of Scaffolds. The micro- and macromorphology of the TPU, HA/TPU, and HA/PDA/TPU scaffolds are shown in Figure 1. It can be seen that a similar 3D network structure with excellent interconnectivity was formed for all TPU, HA/TPU, and PDA/HA/TPU scaffolds. The pore sizes of all samples were approximately 300 μm, which could improve osteogenesis because of higher permeability and potential for vascularization.18 Therefore, it 6383

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Figure 2. XPS spectra of (a, d) TPU, (b, e) HA/TPU, and (c, f) scaffolds. (a−c) PDA/HA/TPU widescan spectra and (b, e, f) C 1s spectra.

were found and could be attributed to O 1s, N 1s, and C 1s, respectively. In comparison with the TPU scaffold, the additional peaks at 348.0 and 136.0 eV could be attributed to the Ca 2p and P 2p, respectively, from the HA particles for the HA/TPU and PDA/HA/TPU scaffolds. This further demonstrated that the HA particles were successfully adhered to the TPU to form the HA/TPU and PDA/HA/TPU scaffolds. However, the peaks intensities that Ca 2p and P 2p exhibited were a little weak due to their lower HA particle content.20,21 From Figure 2d−f, further observation of the C 1s spectra showed that for all TPU, HA/TPU, and PDA/HA/ TPU scaffolds, three peaks attributed to C−C, C−O, and O C−N were observed.22 Furthermore, for the PDA/HA/TPU scaffold, additional peaks at 285.19 and 290.89 eV corresponded to the ketone (CO) and π−π* groups formed by the conjugation effect between the PDA and the HA/TPU matrix.23 From these results, it can be concluded that this conjugation effect occurred, resulting in an increase in the interfacial binding force of the 3-D PDA/HA/TPU scaffold. The XPS atomic compositions of TPU, HA/TPU, and PDA/HA/TPU scaffolds are summarized in Table 1. It can be seen that the O 1s content increased, whereas the C 1s and N 1s contents decreased from TPU to HA/TPU to PDA/HA/ TPU scaffolds. For the HA/TPU and PDA/HA/TPU

Table 1. XPS Atomic Composition of TPU, HA/TPU, and PDA/HA/TPU Scaffolds XPS

C 1s

O 1s

N 1s

P 2p

Ca 2p

others

TPU HA/TPU PDA/HA/TPU

92.04 81.08 77.48

3.57 14.2 18.12

4.35 3.69 3.34

0.21 0.19

0.13 0.15

0.04 0.69 0.71

scaffolds, additional Ca 2p and P 2p compositions existed in comparison with the TPU scaffolds. 2.3. Hydrophilicity and Water Absorption of Scaffolds. Both hydrophilicity and water absorption are dominant features for 3-D tissue engineering scaffolds.24 Figure 3 provides the hydrophilicity and water absorption of TPU, HA/TPU, and PDA/HA/TPU scaffolds. In Figure 3a, the water contact angles decreased from 127.1 to 58.2 and 50.1° for TPU and HA/TPU and PDA/HA/TPU scaffolds, respectively. This indicated that the 3-D scaffolds became more hydrophilic after the addition of HA and PDA particles because many hydroxyls from the HA and PDA particles existed on the surface of the scaffolds.25 Other studies found that the water contact angles ranging from 30 to 60° displayed favorable cell adhesion and proliferation results.26 From Figure 3b, it can be seen that, for all scaffolds, the water absorption 6384

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Figure 3. Hydrophilicity (a) and water absorption (b) of TPU, HA/TPU, and PDA/HA/TPU scaffolds.

Figure 4. Mechanical properties of TPU, HA/TPU, and PDA/HA/TPU scaffolds: (a) typical stress−strain curves, (b) compressive modulus and compressive strength at a strain of 50%, (c) cyclic stress−strain curves, (d) schematic diagram of a compression−release test, and (e) recovery rate of scaffolds after different numbers of cycles.

sharply increased at the initial immersion stage (within 0.5 h) and slightly increased at the next immersion time. In addition, the water absorption of the PDA/HA/TPU scaffolds was slightly lower than that of the TPU and HA/TPU scaffolds, which was in accordance with the porosity change of the scaffold, but it still had a high water absorption rate of more than 1100%. This result showed that the porosity of the scaffold was the major influencing factor on water absorption because the higher the porosity, the more water storage space there was, which is in accordance with the results of our previous work.27 2.4. Mechanical Properties of Scaffolds. The mechanical properties of tissue engineering scaffolds need to satisfy the requirements of the tissue being replaced. Many synthetic

materials have attracted attention due to their exceptional advantages of high strength and stiffness.28−30 Moreover, most human tissues, e.g., heart, skin, blood vessels, liver, muscle, cartilage, and ligaments, also need to have excellent elastic properties that can enable it to bear repeated and dynamic loads and provide shape and stability.31 Figure 4a,b shows the representative compression stress−strain curves of the TPU, HA/TPU, and PDA/HA/TPU scaffolds and the corresponding compressive strengths and compressive modulus at a strain of 50%. It can be found that both the compressive modulus and compressive strength of the HA/TPU and PDA/HA/TPU scaffolds were higher than those of the TPU scaffolds. Moreover, the compressive modulus and compressive strength both increased about 1.4 times from that of the HA/TPU 6385

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Figure 5. Dynamic mechanical properties of scaffolds from dynamic mechanical analysis (DMA) tests: (a) storage modulus and tan δ versus temperature curves for TPU, HA/TPU, and PDA/HA/TPU scaffolds, (b) tan δ of the HA/TPU scaffold at different temperatures and frequencies, (c) tan δ of the PDA/HA/TPU scaffold at different temperatures and frequencies, and (d) a linear regression plot (logarithm of test frequency versus reciprocal Tg).

about 80% in thickness, whereas the PDA/HA/TPU scaffold only deformed by about 50%. These results suggest that the PDA/HA/TPU scaffold possessed comprehensive mechanical properties, such as high strength, stiffness, and excellent flexibility. The recovery rate relative to the original thickness of the scaffold was applied to study its recoverability. Figure 4e shows that there was no obvious decrease in the recovery rates of the TPU and HA/TPU scaffolds after 10 cycles of loading and unloading. Meanwhile, the PDA/HA/TPU scaffold showed the lowest recovery rate, but it still retained a high recovery rate of more than 83% after 10 cycles. 2.5. Dynamic Mechanical Properties. Figure 5a shows the effects of temperature on the storage modulus (E′) and loss factor (tan δ) for the TPU, HA/TPU, and PDA/HA/TPU scaffolds. The test frequency was 1 Hz. It can be seen that the E′ for all types of scaffolds decreased with increasing temperature and declined sharply at a temperature of −40 to 20 °C. This was because the mobility of the molecular chains increased with increase in temperature, thus resulting in an increase in the elastic modulus of the scaffold. For composite scaffolds, several factors like reinforcing filler type, filler dispersion, interfacial adhesion, and matrix type played an important role in the dynamic mechanical properties. The E′ of both the HA/TPU and PDA/HA/TPU scaffolds was higher than for the neat TPU scaffold. For the HA/PDA scaffold, the HA as a rigid particle hindered the mobility of the molecular chains of the matrix, leading to a higher rigidity of the composite scaffolds. For the PDA/HA/TPU scaffold, the PDA coating as a transitional layer improved the interfacial adhesion between the polymer matrix (TPU) and the bioceramics (HA), leading to an increase in interfacial stress transfer and limiting the movement of the molecular chains to improve the strength and rigidity. Furthermore, π−π stacking and hydrogen

scaffold (20.4 and 10.2 kPa) to the PDA/HA/TPU scaffold (28.39 and 14.2 kPa). A possible explanation for these results may be that the HA particles were uniformly immobilized on the surface of the scaffold, which formed a rigid continual phase and increased the strength of the scaffold. Furthermore, with the PDA particles forming a glue-like transition layer, the conjugation effect occurred between the PDA and HA particles and also between the PDA and TPU matrix due to their unique high adhesive force to all types of materials using their rich catechol and amine function groups, resulting in enhanced interfacial adhesion force between the filler and the polymer matrix. Some research has revealed that the interfacial adhesion of composite biomaterials significantly affected their application performance in vivo.32 The mechanical stability of the TPU, HA/TPU, and PDA/ HA/TPU scaffolds under dynamic environments was also evaluated. The energy dissipated in the loading/unloading cycle could be explained by the hysteresis loss. The cyclic stress−strain curves with 1, 2, 5, and 10 loading/unloading cycles are provided in Figure 4c. For all samples, the hysteresis loops decreased slightly with increasing number of cycles and then reached stabilization, which suggests that all scaffolds had good fatigue resistance. After 10 loading/unloading cycles, the hysteresis loop of the PDA/HA/TPU scaffold was smaller than that of the TPU and HA/TPU scaffolds because of the existence of π−π stacking and hydrogen bonding from the PDA. Figure 4d shows the schematic diagram of the compression− release tests for the HA/TPU and PDA/HA/TPU scaffolds. A weight of 100 g was placed on the top of the scaffolds, then the weight was removed, and the scaffolds rapidly recovered to nearly their initial height, thus indicating their good recoverability. When the loading weight was the same, it could be clearly seen that the HA/TPU scaffold deformed by 6386

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Table 2. Tg at Different Frequencies and Activation Energies (ΔE) of the HA/TPU and PDA/HA/TPU Scaffolds Tg (°C) at varying frequencies samples

1 Hz

10 Hz

50 Hz

100 Hz

slope (K)

R2

ΔE (kJ/mol)

HA/TPU PDA/HA/TPU

0.2 8.5

6.4 14.2

9.4 18.4

15.5 22.7

−24.6 −27.8

0.908 0.964

204 231

Figure 6. Morphology of MC3T3-E1 cultured for (a−c) 3 days and (d−f) 5 days on (a, d) TPU, (b, e) HA/TPU, and (c, f) PDA/HA/TPU scaffolds. Scale bar = 10 μm.

equation: f = A exp(ΔE/RTg), where A and f are a frequency factor and the test frequency, respectively. A regression of a line plot of ln(A) versus 1/Tg can be obtained as shown in Figure 5d, and ΔE can be obtained from the slope of the regression line as shown in Table 2. From Table 2, it can be seen that the Tg value significantly increased as the test frequency increased. When the test frequency was the same, the PDA/HA/TPU scaffold displayed a higher Tg compared to the HA/TPU scaffold. The higher Tg indicated that the free motion among the molecular chains occurred at a higher temperature. The ΔE of the HA/TPU scaffold was obtained as 204 kJ/mol, as expected; the higher ΔE of 231 kJ/mol belonged to the PDA/HA/TPU scaffold. This result implied that, for the HA/TPU scaffold, molecular chain relaxation required less energy due to the poor restricting effect generated by the weak interfacial binding force between the HA particles and the TPU matrix. For the PDA/HA/TPU scaffold, the molecular chain relaxation required more energy because of the strong interfacial binding forces between the PDA/HA particles and the TPU matrix, which restricted the molecular movement. Therefore, it can be concluded that the interfacial interaction of the PDA/HA/TPU scaffold was significantly stronger than that of the HA/TPU scaffold due to the addition of the PDA coating. 2.6. Morphology of MC3T3-E1 on the Scaffold. The morphology of MC3T3-E1 on the TPU, HA/TPU, and PDA/ HA/TPU scaffolds is shown in Figure 6 (arrows show the cells). On the TPU scaffold, MC3T3-E1 showed spherical shapes after culturing for 3 days and displayed extension and ladle morphology after culturing for 5 days. On the HA/TPU scaffold, MC3T3-E1 stretched their filopodia and showed partial spreading after culturing for 5 days. This indicates that HA particles promoted cell attachment and growth capacity, which is in agreement with the results of other studies.35 On the PDA/HA/TPU scaffold, MC3T3-E1 showed complete spreading, with no clear individual cell boundaries, and grew along with the surface of the scaffold. This phenomenon revealed that the scaffold−cell interfacial bioactivity signifi-

bonding effects between molecular interactions resulted in a reduction in the mobility of the molecular chains due to the addition of the PDA. In addition, the damping coefficient (tan δ) can represent the energy dissipation for the deformation or irreversible intermolecular movement of the materials. The temperature at the tan δ peak (maximum value of tan δ) usually corresponds to the glass transition temperature (Tg) of the material. From Figure 5a, it can be seen that the Tg of the PDA/HA/TPU scaffold was higher than those of the TPU and HA/TPU scaffolds. Other studies revealed that the stress concentration and viscoelastic energy dissipation for the materials can significantly influence the value of tan δ.33,34 Moreover, for composite materials, the interfacial adhesion between the two or three phases has a very important influence on tan δ. Good interfacial adhesion results in a smaller tan δ, whereas poor interfacial adhesion results in a larger tan δ. It is noteworthy that the maximum value of tan δ for PDA/HA/TPU composite scaffolds was 0.25, which was lower than that of 0.28 for the HA/TPU composite scaffold. Additionally, the width of the tan δ curve for the PDA/HA/TPU composite scaffold was broader than that of the HA/TPU composite scaffold. These results suggest that the interfacial adhesion of the PDA/HA/ TPU scaffold was obviously enhanced compared to the HA/ TPU scaffold due to the introduction of the PDA coating. The tan δ versus temperature curves at frequencies of 1, 10, 50, and 100 Hz for the HA/TPU and PDA/HA/TPU scaffolds are shown in Figure 5b,c. The Tg of the HA/TPU and PDA/ HA/TPU scaffolds with varying frequencies can be obtained from Figure 5b,c, as shown in Table 2. The term “apparent activation energy (ΔE)” can be used to represent the total amount of energy needed to cause molecular movement, including the segment movements that indirectly reflect molecular interactions. The larger the intermolecular force, the more restricted is the molecular chain mobility, thus resulting in more energy required to develop movement of the molecular chains. The ΔE at a temperature of Tg can be described by the exponential term −ΔE / RTg in the Arrhenius 6387

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excellent comprehensive mechanical properties. In vitro studies showed that the addition of the PDA coating effectively improved the adhesion and viability of MC3T3-E1 cultivated on the PDA/HA/TPU scaffold. These results suggest that the PDA/HA/TPU scaffold possesses great potential to be used as a tissue engineering scaffold, whereas the PDA coating could be a simple, effective, and universal approach to modify composite scaffolds.

cantly improved due to the excellent cell−scaffold bonding between the PDA coating and MC3T3-E1. 2.7. Viability of MC3T3-E1 on the Scaffold. An ((3(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay to quantitatively assess the activity of MC3T3E1 on the TPU, HA/TPU, and PDA/HA/TPU scaffolds is shown in Figure 7. For all scaffolds, the number of cells

4. EXPERIMENTAL METHODS 4.1. Materials. Thermoplastic polyurethane (TPU) (Tecoflex EG-80A, Mw = 201 873 Da, Mn = 94 212 Da) was obtained from Noveon, Inc. Hydroxyapatite (HA, 99%) nanoparticles, with an average diameter of 20 nm, were obtained from Nanjing Emper Nano Material Co, Ltd., China. Sodium chloride particles (NaCl) were obtained from Jiangsu Salt Industry Group Co., Ltd., China. The ethanol, 1,4-dioxane, and tetrahydrofuran were obtained from Guang Zhou Jinhuada Chemical Reagent Co. Ltd., China. Dopamine and tris(hydroxylmethyl) aminomethane (Tris) were supplied by Aladdin. The mouse embryonic osteoblasts cells (MC3T3-E1) were obtained from Shanghai Gefan Biotechnology Co., Ltd., China. 4.2. Preparation of 3-D Porous HA/TPU Scaffolds. To prepare 3-D porous HA/TPU scaffolds, a combination of thermally induced phase-separation, particle-leaching, and ultrasonic-assisted techniques were used. First, TPU pellets were dried for 2 h at 80 °C to remove moisture. Then, 1.6 g of TPU pellets were dissolved in 20 mL of a 1,4-dioxane/water mixture (17:3, v/v) at 70 °C with magnetic stirring for 12 h to obtain a homogeneous TPU solution. A certain amount of sieved NaCl particles with diameters ranging from 225 to 300 μm were placed in a plastic mold and compressed to form the NaCl preform. Then, the NaCl preformed was completely filled and wetted using the TPU solution at 60 °C with the help of the vacuum-assisted technique. For a detailed description of the vacuum-assisted solvent-casting method for the fabrication of 3-D porous scaffolds, please refer to our previous study.36 The prepared TPU/NaCl/1,4-dioxane/water blend was quickly transferred into a −80 °C freezer overnight. Then, the prefrozen blend was immersed in an ethanol bath at −20 °C for 2 days to extract the solvent (1,4-dioxane). After solvent extraction, the prepared NaCl/TPU blend was dried and then leached in deionized water for 2 days to remove the porogen NaCl particles. Second, 70 mg of HA nanoparticles was placed into 70 mL of deionized water and subjected to ultrasonication treatment for 60 min to prepare a homogeneous HA suspension. Then, the 3-D porous TPU scaffold was immersed in an HA suspension and subject to ultrasonication treatment for 30 min. During ultrasonication, when the ultrasonication power was larger enough, the HA nanoparticles were pushed against the TPU scaffold at high speed and immobilized on the surface of the TPU scaffold. For more information on the immobilization mechanism of nanoparticles on the surface of a polymer matrix using the ultrasonication method, please refer to our previous study.37 The HA/TPU scaffold was fabricated and washed several times to remove the unanchored HA nanoparticles and dried in a vacuum oven for 24 h. 4.3. Preparation of 3-D Porous PDA/HA/TPU Scaffolds. The HA/TPU scaffolds were placed into a 2 mg/mL dopamine solution prepared using 10 mM Tris−HCl buffer

Figure 7. Cell viability of MC3T3-E1 on TPU, HA/TPU, and PDA/ HA/TPU scaffolds after culturing for 1, 3, 5, and 7 days. *p < 0.05, **p < 0.01, and ***p < 0.001 with n = 8. All error bars presented are mean ± standard deviation.

increased with increasing culture time. After culturing for 3 days, the number of cells on the PDA/HA/TPU scaffold was obviously higher than that on the TPU scaffold. Meanwhile, after culturing for 5 and 7 days, the number of cells on the PDA/HA/TPU scaffold was significantly greater than that on both the TPU and HA/TPU scaffolds. For example, the viability of MC3T3-E1 on the PDA/HA/TPU scaffold was 5.3 times and 3.2 times higher than on the TPU and HA/TPU scaffolds after culturing for 7 days, respectively. This result implies that the PDA coating was highly effective in increasing the cell adhesion and viability on the scaffolds, and its enhancement effect was even higher than that of the HA particles. These results might be attributable to the following facts: (1) the PDA coating improved the hydrophilicity of the scaffold, resulting in excellent attachment of MC3T3-E1 on the surface of the scaffold; and (2) the unique strong adhesion properties of the PDA coating strengthened the interaction between the cells and the scaffold.

3. CONCLUSIONS In summary, PDA was successfully coated onto the HA/TPU scaffold by dopamine self-polymerization. The morphology, water absorption, porosity, hydrophilicity, mechanical properties, and cell viability of the scaffolds were systematically studied. The water contact angles decreased from 127.1 to 58.2° for the TPU scaffold and to 50.1° for the PDA/HA/TPU scaffold, which indicated that the PDA/HA/TPU scaffold had excellent hydrophilicity. Both the compressive modulus and the compressive strength of the PDA/HA/TPU scaffold increased about 1.4 times in comparison with that of the HA/TPU scaffold. In comparison with the HA/TPU scaffold, although the strength and stiffness of the PDA/HA/TPU scaffold was improved, excellent elasticity was retained with a high recovery rate of more than 83%. Furthermore, DMA revealed that the interfacial binding force between the HA particles and the TPU matrix was significantly improved by the PDA coating. Hence, the PDA/HA/TPU scaffold possessed 6388

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Figure 8. Schematic of composite scaffold preparation.

4.8. Static-State Mechanical Testing. The compressive mechanical properties of the TPU, HA/TPU, and PDA/HA/ TPU scaffolds were studied using a universal testing machine (UTM2102 Suns Technology Stock Co. Ltd, China). All samples were circular disks with a height of 10 mm and a diameter of 10 mm. A crosshead speed of 3 mm/min was applied at room temperature and the maximum compressive strain was set as 50%. For each group, eight samples were measured. The compressive modulus was obtained from the slope of the straight line portion of the stress−strain curve. The compressive strength was calculated at a strain of 50%. The samples were compressed and then released repeatedly at a strain of 50% for 10 cycles to observe their cyclic properties. Equation 3 was applied to calculate the shape recovery rate in each cycle39

(pH 8.5) and stirred for 24 h at room temperature. A PDA coating layer was generated on the surface of the 3-D porous HA/TPU scaffold. Then, the fabricated PDA/HA/TPU scaffold was thoroughly washed with deionized water and ethanol 3−5 times to remove the nonadhered PDA particles and dried in a vacuum oven at 50 °C for 24 h. The schematic of the process for fabricating a PDA/HA/TPU scaffold is shown in Figure 8. 4.4. Morphology of 3-D Porous Scaffolds. The microstructures of the samples were observed using a scanning electron microscopy (SEM) (Nova NanoSEM 450, FEI Company, Hillsboro, OR). Before examination, the scaffold was brittle-fractured in a liquid nitrogen bath and then its cross section was sputtered with a gold coating (Ace200, Leica, Germany) for 30 s. 4.5. Porosity of 3-D Porous Scaffolds. The porosity of the scaffold was measured via a solvent-replacement method.38 A dried scaffold was weighed and recorded as M1 and then placed into an ethanol solution until its weight no longer changed. The wet scaffold was weighed and recorded as M2. Equation 1 was used to calculate the porosity of the scaffold porosity = (M 2 − M1)/ρV

l Lunloading | o o o o − recovery rate = m 1 } × 100% o o o L loading o o o n ~

where Lloading and Lunloading are the strains when the stress increased from zero or decreased to zero in the loading and unloading cycle, respectively. 4.9. Dynamic Mechanical Analysis. Dynamic mechanical analysis (DMA 242E, Netzsch, Germany) was used to study the storage modulus and glass transition temperature (Tg) of the scaffolds. All samples were circular disks with a diameter of 10 mm and a height of 6 mm. The tests were carried out in compression mode at frequencies of 1, 10, 50, and 100 Hz. The heating rate was 2 °C/min between −100 and 100 °C. 4.10. Evaluation of Scaffolds in Vitro. MC3T3-E1 were used for cell viability and cell proliferation studies. The 3-D porous TPU, TPU/HA, and TPU/HA-PDA scaffolds were sterilized by putting them in 70% ethanol for 24 h and then exposing them to UV light for 12 h on each side. The cell viability was assessed using an MTT assay ((3-(4,5)dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide). MC3T3-E1 were seeded at a density of 1000 cells/mL in a 96-well plate and incubated for 1, 3, 5, and 7 days at 37 °C with 5% CO2 at 95% humidity. After a certain culture time, 20 μL of MTT solution (5 mg/mL diluted in fresh Roswell Park Memorial Institute-1640 medium) was placed in the culture plate and cultured for 4 h at 37 °C. Afterward, 200 μL of dimethyl sulfoxide was added to solubilize the formazan salts under stirring. Finally, the mixture was analyzed using a microplate reader 450 (Bio-Rad instrument) at 570 nm. Quantitative data were obtained for eight samples (n = 8) and reported with a mean standard deviation. An analysis of variance with multiple post hoc comparisons was performed using Tukey’s test with values of p < 0.05 (*) considered statistically significant, whereas p < 0.01 (**) considered extraordinarily significant.

(1)

where ρ is the density of the ethanol solution and V is the volume of the scaffold. 4.6. X-ray Photoelectron Spectroscopy. X-ray photoelectron spectroscopy (XPS, VGS ESCALAB 250A) was used to analyze the chemical compositions of the TPU, HA/TPU, and PDA/HA/TPU scaffolds. The pressure of the chamber was 10−8 torr and the take-off angle was 45°. The binding energies were calibrated using the containment C 1s hydrocarbon peak at 284.6 eV. 4.7. Hydrophilicity and Water Absorption of 3-D Porous Scaffolds. Water contact angle measurements were performed using a contact angle goniometer (DSA 25, KRUSS, Germany). The samples were attached to coverslips and 2.5 μL of deionized water was dripped onto the surface of the scaffold. Five different positions on each sample were measured. The water absorption was performed by testing the water uptake ability of the scaffold in phosphate-buffered saline (PBS) (pH = 7.4) at 37 °C. The samples were immersed in the PBS solution, and the scaffolds were taken out at regular time intervals. The surface water on the scaffold was removed by filter paper and then the scaffold weight was recorded. The water absorption (%) of the scaffold was determined using eq 2. The water absorption (%) of the scaffold was obtained for 10 samples and reported as a mean standard deviation water absorption (%) = (Ws − Wi )/Wi × 100%

(3)

(2)

where Ws represents the swollen scaffold weight and Wi represents the initial scaffold weight. 6389

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The MC3T3-E1 morphology cultured on the 3-D porous scaffolds was observed by SEM. Before SEM observation, the cultured cells were fixed at 4 °C using 4% paraformaldehyde for 8 h and then dehydrated via gradient ethanol. The dehydration time for each ethanol content (50, 70, 80, 90, 95, and 100%) was 10 min. The cross sections of the 3-D porous scaffolds were observed via SEM.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (Z.C.). *E-mail: [email protected] (L.-S.T.). ORCID

Zhixiang Cui: 0000-0001-9725-6181 Lih-Sheng Turng: 0000-0001-8022-9224 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the financial support of the Outstanding Young Scientific Research Personnel Training Plan in Colleges and Universities of Fujian Province (Grant no. GY-Z160146), the Research Fund of Fujian University of Technology (Grant nos. GY-Z15091, GY-Z160121), The Program of New Century Excellent Talents in University of Fujian Province (GY-Z17065), the Research Fund for Industry-University Cooperation of Fujian Province (Grant no. 2016H6001), the National Natural Science Foundation of China (Grant no. 51303027), and Special fund for visiting scholars in Jiangxi province’s development plan for young and middle-aged teachers in ordinary undergraduate universities.

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