Dopamine-incorporated Dual Bioactive Electroactive Shape Memory

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Dopamine-incorporated Dual Bioactive Electroactive Shape Memory Polyurethane Elastomers with Physiological Shape Recovery Temperature, High Stretchability and Enhanced C2C12 Myogenic Differentiation Xin Zhao, Ruonan Dong, Baolin Guo, and Peter X Ma ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b10583 • Publication Date (Web): 16 Aug 2017 Downloaded from http://pubs.acs.org on August 17, 2017

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ACS Applied Materials & Interfaces

Dopamine-incorporated

Dual

Bioactive

Electroactive

Shape

Memory

Polyurethane Elastomers with Physiological Shape Recovery Temperature, High Stretchability and Enhanced C2C12 Myogenic Differentiation

Xin Zhao a†, Ruonan Dong a†, Baolin Guo a,*, Peter X. Ma a,b,c,d,e,* a

Frontier Institute of Science and Technology, and State Key Laboratory for

Mechanical Behavior of Materials, Xi’an Jiaotong University, Xi’an, 710049, China b

Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI

48109, USA c

Department of Biologic and Materials Sciences, University of Michigan, Ann Arbor,

MI 48109, USA d

Macromolecular Science and Engineering Center, University of Michigan, Ann

Arbor, MI 48109, USA e

Department of Materials Science and Engineering, University of Michigan, Ann

Arbor, MI 48109, USA * To whom correspondence should be addressed. Tel.:+86-29-83395317. Fax: +86-29-83395131. E-mail: [email protected], [email protected]. † These authors contributed equally to this work.

1

ACS Paragon Plus Environment


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Abstract: Soft tissue engineering needs elastic biomaterials not only mimicking the elasticity of soft tissue but also possessing multiple bioactivity to promote cell adhesion, proliferation and differentiation, which still remains ongoing challenges. Herein, we synthesized a series of dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers by combining the properties of elastomeric poly(citric acid-co-polycaprolactone) (CA-PCL) polyurethane elastomer, bioactive dopamine (DA) and electroactive aniline hexamer (AH). The chemical structures, electroactivity, conductivity, thermal properties, hydrophilicity and hydration ability, mechanical properties, and degradability of the polyurethane elastomers were systematically characterized. The elastomers showed excellent shape fixity ratio and shape recovery ability under physiological conditions. The elastomers’ elongation and stress were tailored by AH content, while the hydrophilicity and hydration ability of the elastomers were adjusted by content of DA and AH, as well as the doping state of AH. The viability and proliferation results of C2C12 cells seeded on the elastomers showed their excellent cytocompatibility. Additionally, by analyzing the protein and gene level, the promotion effect on myogenic differentiation of C2C12 cells by these elastomers compared to control groups (PCL80000, CA-PCL elastomer, and CA-PCL elastomer with DA segment) was demonstrated. Furthermore, the results from subcutaneous implantation confirmed the elastomers’ mild host response in vivo. These results represent that these dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers are promising candidates for soft tissue regeneration that is sensitive to electrical signal. 2


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Keywords: Dopamine-incorporated biomaterials, shape memory elastomers, aniline oligomer, electroactivity, dual bioactivity, myogenic differentiation

1 Introduction Most of the human native tissues, especially for soft tissues such as cardiac muscle and skeletal muscle, present highly elastic properties (resilience and stretchability).1 Synthetic biodegradable elastomers have attracted significant attention in soft tissue regeneration applications for their unique features including 3D crosslinked networks mimicking the structure of naturally-derived elastic materials, high elasticity and flexibility, biodegradability, as well as mechanical properties similar to those of native soft tissues.2-3 Poly(ε-caprolactone) (PCL), a synthetic thermoplastic polyester elastomer with good biocompatibility, widespread usage, and low cost has been widely used in soft tissue engineering,4-6 such as cardiac tissue engineering,7 muscle tissue engineering,8 nerve tissue engineering,9 bladder tissue engineering,10 and etc.. However, its mechanical characteristics dissatisfy the native soft tissues’ requests due to its plastic deformation and failure when exposed to long-term cyclic strain, limiting their performance in soft tissue engineering applications5, 11. In the past decades, a series of biodegradable polyester elastomers were developed via melting polycondensation of polybasic carboxylic acid and polyalcohol, and they have received much attention in soft tissue engineering, especially, citric acid is polyorganic acid existing in human body and widely used to synthesize polyester pre-polymer with polyalcohols via melting polycondensation reaction3, 3


12-14

. Thus,


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melting polycondensation of low molecular polycaprolactone diols (PCL) and citric acid will combine the characteristic properties of PCL and poly(polyhydric alcohol-co-polyatomic acid) to develop good candidate materials for soft tissue engineering. Moreover, much different from polyester elastomers such as PGS and POC without Tm and with Tg below 0 oC, the introduction of semi-crystalline PCL segment into the polyester elastomer will endow the material with adjustable Tm for shape memory capacity15. The adjustable Tm of the polyester elastomer also can endow the material with good elastic properties under physiological conditions and rapid physiological conditions induced shape memory capability16-19.

The

physiological conditions induced shape memory capability can drive the materials to rapidly self-expand to their original shape under body temperature without external stimulus, which allows the materials to be transplanted into the human body via minimally invasive surgery20-22. Besides, the shape memory property of the materials can also be used in combination with muscle cells for muscle transfer for plastic surgery and tissue reconstruction etc.23. Interestingly, our previous report used three PCL diols (Mn=2000, 3000, and 4000 Da, respectively) to synthesize shape memory polyurethane copolymers, and we found that using PCL diols with a molecular weight of 2000 Da could synthesize the shape memory copolymer with slightly lower Tm than body temperature and much lower Tc than 0 oC, which inspired us to employ PCL2000 to synthesize shape memory polyester elastomer with physiological shape recovery temperature and good resilience for their rubbery states and amorphous structure under body temperature24. As smart stimulus-responsive materials, 4


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biodegradable shape memory elastomers with rapid physiological switching property and good resilience under physiological conditions will be highly desired in biomedical applications. Synthetic polyester elastomers including PCL, PGS and POC generally presented poor hydrophilicity, limited hydration ability and low bioactivity1, 25-27. To address this issue, physically blending elastomers with bioactive components, chemically grafting bioactive components into elastomers’ networks, as well as functionalizing elastomers surface with bioactive components to improve the materials’ bioactivity are developed1, 5, 8-9, 25-29. Among the above approaches, chemically grafting bioactive molecular into elastomer, endowing the material with intrinsic activity, is a promising way in this field. In recent years, polydopamine modified biomaterials showed increasing application in tissue engineering for their highly bioactive catechol groups, which can interact with cells and biomacromolecules containing thiol- or aminegroups to enhance the materials’ activity29-30. Similarly, DA with reactive catechol group to absorb amine- or thiol-contained biomacromolecules via Schiff-base or Michael addition chemistry has also been demonstrated to support cell adhesion and promote cell proliferation29-33. However, as to chemically grafting way to improve elastomer’s bioactivity, dopamine functionalized elastomer used for tissue engineering has not been reported. We hypothesized that dopamine can be introduced into poly(polycaprolactone-co-citric acid) elastomer during melting polycondensation to synthesize

dopamine-functionalized

poly(polycaprolactone-co-citric

acid-co-dopamine) polyester elastomers. 5



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Electroactivity of materials is also a vital property that could affect cell behavior, especially for the electrical signal sensitive cells, such as cardiac myoblast, skeletal myoblast, nerve cell and etc.34-43, which proposes the scaffolds with electroactivity to enhance the specific cell responses. Recently, aniline oligomers with good biocompatibility, processability, degradability and electroactivity draw more and more attention in electroactive scaffold preparation44-53. Our group have developed a series of electroactive biomaterials using aniline oligomers including aniline trimer, aniline tetramer and aniline pentamer, and demonstrated the positive effect of aniline oligomers’ electroactivity on electrical signal sensitive cells’ adhesion, proliferation and differentiation24, 35, 38-39, 54. Thus, introducing electroactive aniline oligomer into dopamine-functionalized polyurethane elastomer will result in dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers. As most of the reported materials for myogenic differentiation usually presented low bioactivity or just had a single bioactivity24, 35, 39, 55-57, we hypothesize that dual bioactive shape memory polyurethane elastomers simultaneously possessing bioactive catechol group and electroactivity will synergistically promote the myogenic differentiation, which will provide a new view to develop materials for myogenic differentiation applications. Moreover, compared with the reported shape memory electroactive materials with recovery temperature higher than body temperature for myogenic differentiation24, the dual bioactive shape memory polyurethane elastomers presenting shape recovery capability and good resilience under physiological conditions will be highly anticipated in electrical signal sensitive tissues’ regeneration such as skeletal 6


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muscle repair. However, it is still a challenge. Herein, we developed a series of dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers based on hexamethylene diisocyanate (HDI) crosslinked

poly(citric

acid-co-polycaprolatone-co-dopamine)

(CA-PCL-DA)

pre-polymer and aniline hexamer (AH) to significantly enhance C2C12 cell’s adhesion, proliferation and myogenic differentiation for skeletal muscle regeneration. Firstly, HDI crosslinked CA-PCL pre-polymer elastomer was synthesized as the basic material for its good biocompatibility, high elasticity and biodegradable property. Then, different amount of DA was incorporated into CA-PCL elastomer resulting in DA functionalized CA-PCL-DA elastomers. DA functionalized CA-PCL-DA elastomer with optimized molar ratio of CA:PCL:DA=1:1:0.3 to enhance C2C12 cell’s adhesion and proliferation was further used to fabricate dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers by introducing different content of electroactive AH into the CA-PCL-DA elastomer. The molecular structures, electroactivity, conductivity, thermal properties, hydrophilicity and hydration ability, mechanical properties, biodegradability and thermally induced shape memory capability of the synthesized elastomers were comprehensively investigated. The electroactive elastomers with different AH content present tunable stress,

tunable

wettability,

tunable

modulus,

excellent

stretchability,

and

biodegradability. Moreover, the elastomers presented ∼100% shape fixity ratio under 0 oC, and recovered in a short time under physiological conditions. Furthermore, both the introduction of DA and AH into CA-PCL elastomer could promote C2C12 cell 7



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adhesion, proliferation and myogenic differentiation, and all of the elastomers just showed mild host response when transplanted subcutaneously in vivo. All these data suggested that it is a promising way to develop physiological conditions induced shape memory polyurethane elastomer via one-pot melting polycondensation using semi-crystalline polyester segment (e.g. PCL) and polybasic carboxylic acid (e.g. CA), and further endow the shape memory polyurethane elastomers with dual bioactivity by

incorporation

of

DA and

electroactive

AH

segment.

The

resultant

dopamine-incorporated dual bioactive electroactive shape memory polyurethane elastomers perform great potential for electrical signal sensitive soft tissue regeneration applications. 2 Materials and methods 2.1 Materials Citric acid (CA), polycaprolactone diols (PCL) (Mn = 2000), poly(ε-caprolactone) (Mn = 80000), stannous octoate (Sn(Oct)2), N, N-Diphenyl-p-phenylenediamine, hexamethylene diisocyanate (HDI), ammonium persulfate and p-phenylenediamine were purchased by Sigma-Aldrich. Dopamine (DA) hydrochloride and anhydrous N, N-dimethylformamide (DMF) were purchased from J&K Scientific Ltd. Aniline was distilled twice before use and other reagents were analytic grade. 2.2 Synthesis of poly(citric acid-co-polycaprolactone-co-dopamine) pre-polymer The poly(citric acid-co-polycaprolactone-co-dopamine) (CA-PCL-DA) pre-polymer was synthesized via melting polycondensation reaction. In brief, citric acid (CA) and polycaprolactone diols (PCL) (Mn = 2000) were added to a three-necked 8


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round-bottom flask, and then heated to 160 °C to obtain a clear molten mixture under nitrogen environment with stirring. Following that, different amount of dopamine was introduced into the molten mixture under nitrogen gas flow. After obtaining a clear mixture when heating at 160 oC, the heating temperature was cooled down to 140 oC and the pressure was reduced to 5 kPa under vacuum for required time to obtain polyester pre-polymer with desired viscosity. After the reaction was finished, the product was dissolved in dichloromethane, filtrated and then precipitated in precooled diethyl ether to purify the product. The dissolution-filtration-precipitation process was repeated for three times to obtain purified pre-polymer. Three pre-polymers containing different molar ratios of dopamine (molar ratios of CA:PCL:DA varying from 1.0:1.0:0.0, 1.0:1.0:0.3 and 1.0:1.0:0.5) were synthesized in this study. 2.3 Synthesis of electroactive aniline hexamer (AH) The electroactive aniline hexamer (AH) was synthesized by two steps. Firstly, amine-capped aniline trimer was synthesized according to a previous report58. Then, aniline hexamer (AH) was synthesized using amine-capped aniline trimer and N, N-Diphenyl-p-phenylenediamine as previously described59. The details are available in SI Materials and methods. 2.4 Preparation of CA-PCL-DA elastomer and electroactive CA-PCL-DA-g-AH elastomer The

CA-PCL-DA

elastomer

and

acid-co-polycaprolactone-co-dopamine)-g-aniline

electroactive hexamer

poly(citric

(CA-PCL-DA-g-AH)

elastomers with different amount of AH were prepared using HDI as crosslinker. In 9



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brief, 0.8 g of CA-PCL-DA pre-polymer was dissolved in 4 mL of anhydrous DMF, and different amount of AH (varying the weight ratios of AH in CA-PCL-DA and AH mixtures from 0 wt%, 3 wt%, 6 wt% and 9 wt%) was dissolved in 8 mL of anhydrous DMF, respectively. Then, desired amount of HDI was added into the corresponding AH solution with Sn(Oct)2 (0.15 wt%) as catalyst, while keeping 0.5:1.0 molar ratio of HDI to the residual hydroxyl groups from CA-PCL-DA and amino groups from AH. After vigorously stirred for 4 h at room temperature, the HDI or AH/HDI reactant was poured into the corresponding CA-PCL-DA pre-polymer. The mixture was stirred at 55 oC for 10 min, poured into a teflon dish and placed at oven at 55 oC for 12 h to obtain crosslinked CA-PCL-DA network or crosslinked CA-PCL-DA-g-AH network. Then, the solvent was completely evaporated at 55 oC for another 24 h. For the camphorsulfonic acid (CSA) doped electroactive CA-PCL-DA-g-AH elastomers, AH and CSA (molar ratio of quinoid units in AH to CSA was 1:2) were simultaneously dissolved in DMF, and other procedure was the same with the above described. 2.5 Characterizations The nuclear magnetic resonance (1H NMR), Fourier transform infrared spectroscopy (FTIR), gel permeation chromatography (GPC), UV-vis spectroscopy, cyclic voltammetry (CV), conductivity test, degradation test and scanning electron microscope (SEM) were used to investigate the chemical and physical characterizations of pre-polymers or elastomers. The details are available in SI Materials and methods. 2.6 Hydrophilicity and hydration ability of the electroactive elastomers 10


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The elastomers’ hydrophilicity was tested using water contact angle measurement (SL200KB, Kono), while the hydration kinetic study of the elastomer was performed by testing the water uptake profile of the elastomer. The details are available in SI Materials and methods. 2.7 Thermal properties of electroactive elastomers The thermal physical properties of the elastomer including melting temperature (Tm), crystallization temperature (Tc) and crystallinity (Xc) were measured by differential scanning calorimetry (DSC, DSC-2920, TA). The details are available in SI Materials and methods. 2.8 Mechanical properties of the electroactive elastomers The mechanical properties of the elastomers were evaluated by uniaxial tensile test and cyclic tensile test employing an instron Materials Test system (MTS Criterion 43, MTS Criterion) equipped with a 50 N load cell. The details are available in SI Materials and methods. 2.9 Shape memory behavior in water Thermally induced shape memory capability of the elastomers in water baths with temperatures of 20 °C, 37 °C and 42 oC, was investigated by following the previous reports17, 60. The details are available in SI Materials and methods. 2.10 Dynamic mechanical analysis The

dynamic

mechanical

properties

of

the

CA-PCL,

CA-PCL-DA

and

CA-PCL-DA-g-AH elastomers were tested using a Q800 DMA (TA Instruments) equipped with tensile film clamps. The sample specimens with dimensions of 30.0 11



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mm × 6.0 mm × 0.2 mm were used and the temperature was ramped from 0 °C to 100 °C at a heating rate of 2.0 °C/min. The strain amplitude and frequency were 1% strain and 1.0 Hz, respectively 61. 2.11 Stress-controlled cyclic thermal mechanical test The stress-controlled cyclic thermal mechanical test according to reference61 was performed using Q800 DMA employing a tensile fixture to evaluate the shape memory capability of the elastomers. The details are available in SI Materials and methods. 2.12 Cultivation of C2C12 cells and seeding on the elastomers C2C12 cells were purchased from ATCC (American Type Culture Collection) and the details of cultivation of C2C12 cells and seeding on the elastomers are available in SI Materials and methods. 2.13 C2C12 cells viability and proliferation on the elastomers Live/Dead assay and Alamar Blue assay were used to measure the cell viability and cell proliferation on the elastomers, respectively. The details are available in SI Materials and methods. 2.14 Myogenic differentiation of C2C12 cells The details are available in SI Materials and methods. 2.15 Immunofluorescence The details are available in SI Materials and methods. 2.16 qRT-PCR Expression of two of the specific myogenesis genes, Myogenin (MyoG) and Troponin 12


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T (TnnT), along with housekeeping gene glutaraldehyde phosphate dehydrogenase (GAPDH), were assessed to verify the myogenic differentiation of the myoblasts using the qRT-PCR. The details are available in SI Materials and methods. 2.17 In vivo host response of elastomers All the procedures of animal experiments were performed according to the guidelines established by the committee on animal research at Xi'an Jiaotong University. Before implantation, all the materials were cut into the same shape and size (10 mm × 3 mm × 0.2 mm), sterilized with 75% ethanol and rinse in PBS overnight. Female Sprague Dawley (SD) rats, 200-250 g in weight, were generally anesthetized and a small incision was made on the same area on the back of each rat. The testing articles were placed subcutaneously into the incision and the skin was closed following the placement. The rats returned to their own cage and permitted free access to food and water after they recovered from anesthesia. 2 weeks after surgery, the animals were sacrificed and implanted materials were excised with the adjacent tissues. After excision, the material-tissue compounds were embedded in paraffin, sectioned (3 µm), mounted onto slides. Acute inflammatory response was evaluated from both hematoxylin and eosin (H&E) and toluidine blue (TB) staining. After staining, the slides were observed under 400× microscope and the images were analyzed using Image Pro Plus (IPP) software. 2.18 Statistic analysis All the data from the study were presented as mean ± standard deviation (SD) and analyzed using Student's t test. P value < 0.05 was considered as statistical difference. 13



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3 Results and discussions 3.1 Synthesis of CA-PCL-DA pre-polymers and electroactive CA-PCL-DA-g-AH flexible polyurethane elastomers CA-PCL-DA pre-polymers were synthesized by melting polycondensation of citric acid (CA), polycaprolactone diols (PCL) (Mn = 2000) and dopamine (DA) (Figure 1a). Three pre-polymers were synthesized by changing the molar ratios of CA: PCL: DA varying from 1:1:0, 1:1:0.3 to 1:1:0.5. The samples were coded as CA-PCL pre-polymer, CA-PCL-DA0.3 pre-polymer and CA-PCL-DA0.5 pre-polymer, respectively. In order to choose an optimal DA content for cell culture, we firstly studied the effect of DA content in the three HDI crosslinked pre-polymer elastomers (coded as CA-PCL elastomer, CA-PCL-DA0.3 elastomer, and CA-PCL-DA0.5 elastomer) on adhesion, viability and proliferation of C2C12 cells. As shown in Figure S1a and Figure S1b, compared with PCL80000 and CA-PCL elastomer, both CA-PCL-DA0.3 elastomer and CA-PCL-DA0.5 elastomer presented higher cell adhesion, viability and proliferation (p 25 oC). However, the other HDI crosslinked polyurethane elastomers showed near-liner tensile stress–strain curves without obvious yielding phenomenon for their much lower Tc (-19.5 oC ~ -13.2 oC) than PCL80000. Furthermore, CA-PCL elastomer had elongation of 450%, stress of 15.2 MPa and modulus of 9.0 MPa. However, when introducing DA into CA-PCL elastomer, CA-PCL-DA0.3 elastomer had higher elongation (550%), stress (21.2 MPa) and modulus (13.7 MPa) due to the physical interactions between catechol groups including π-π stacking and hydrogen bonding. When introducing AH content into the CA-PCL-DA0.3 elastomer and varying the AH content from 3 wt% to 9 wt%, both the elongation, stress and modulus of the electroactive elastomers continuously decreased from 550% to 360%, from 21.2 MPa to 10.6 MPa, and from 13.7 MPa to 26


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3.6 MPa, respectively. This was because that more AH would consume more free hydroxyl groups from CA-PCL-DA0.3 pre-polymer leading to less chemical crosslinking between hydroxyl groups and HDI in the CA-PCL-DA-g-AH elastomers. Furthermore, the hard segment of AH would further reduce the elongation of the CA-PCL-DA-g-AH elastomers for their strong physical interactions24, 38. In this study, a series of CA-PCL-DA-g-AH electroactive polyurethane elastomers with tunable mechanical properties (modulus of 3.6-13.7 MPa and elongation of 360-550%) were prepared by changing AH content in polyurethane network, and these results suggested that the CA-PCL-DA-g-AH elastomers might present wide potential applications for soft tissue engineering. The mechanical stability of the elastomers CA-PCL, CA-PCL-DA0.3 and CA-PCL-DA-g-AH3 under dynamic environments was also evaluated. Fifty successive loading–unloading cycles were performed. CA-PCL elastomer showed the smallest hysteresis loop for its covalent crosslinking among the polymeric chains and lowest crystallinity at the test temperature. However, with the introduction of AH and /or DA into CA-PCL elastomer, they showed gradually enhanced hysteresis loops from CA-PCL-DA0.3 elastomer to CA-PCL-DA-g-AH3 after first loading–unloading cycle for the existence of physical interactions (π-π stacking and hydrogen bonding from AH and DA). While from the second cycle to the fifty cycle, all the three samples exhibited a very small hysteresis loop (Figure 4(c-e)), minimal loss of tensile strength and minimal creep deformation, suggesting the good fatigue resistance of all the elastomers. 27



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Figure 4. Mechanical properties of the elastomers. (a) Stress–strain behavior. (b) Summary of mechanical properties of the elastomers. Hysteresis loops of CA-PCL elastomer (c), CA-PCL-DA0.3 elastomer (d) and CA-PCL-DA-g-AH3 (e) at 1st, 10th, 20th, 30th, 40th, and 50th cycle under 50 cyclic tensile tests. 3.7 Shape memory capacity of the elastomers The prepared elastomers presented Tm from 29.4 oC - 35.2 oC slightly lower than human body temperature. Thus, these elastomers might show good shape-memory capacity, and the good shape memory property of these elastomers is beneficial for their invasive surgery for skeletal muscle regeneration. We first evaluated their shape memory ability qualitatively and quantitatively using water baths (20 oC, 37 oC, and 42 oC, respectively). As shown in Figure 5, all the film specimens showed excellent shape fixity ability in ice-bath (Figure 5b) except for PCL80000 showing partial shape fixity. Furthermore, when placing the film specimens into 37 oC water bath, PCL80000 just partly recovered its shape, while the other films almost recovered to 28


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their initial shapes (Figure 5c) revealing their excellent shape recovery properties under body temperature. We also wrapped CA-PCL elastomer, CA-PCL-DA0.3 elastomer, and CA-PCL-DA-g-AH3 elastomer around a cylinder at a sufficiently high temperature (42 °C) for 5 min, then the elastomers were cooled using an ice-bath for 5 min to obtain the temporary spiral shapes (Figure 5d). These spiral shapes kept stable in 20 oC water bath and rapidly recovered to their initial shapes when immersed in 37 o

C water bath (Figure 5d) indicating their capacity to form complicated and stable

temporary shapes for biomedical applications. Figure 5e showed the quantitative results of shape memory behavior of the elastomers, which were deformed at 42 oC and recovered at 20 oC, 37 oC, and 42 oC, respectively. All the elastomers showed 100% fixity ratio (Rf) when deformed at 42 oC and cooled at 0 oC, except for PCL80000 (Rf ≈ 75% at 42 oC) due to its higher Tm (58.6 oC) than 42 oC. PCL80000 film showed slight recovery ratio (Rr) of 5%, while the other elastomers showed 0 % recovery at 20 oC. When increasing the recovery temperature to 37 oC or 42 oC, PCL80000 showed Rr=47% and Rr=53% at 37 oC and 42 oC, respectively. While both CA-PCL elastomer and CA-PCL-DA0.3 elastomer showed Rr of 100% within several seconds at 37

o

C and 42

o

C. With the introduction and increase of AH content in

CA-PCL-DA0.3 elastomer, the Rr of the electroactive elastomers varied from 99% for CA-PCL-DA-g-AH3, 94% for CA-PCL-DA-g-AH6, to 92% for CA-CPL-DA-g-AH9 at 37 oC, and the recovery time of the electroactive elastomers were between 46 s and 58 s. When increasing the recovery temperature to 42 oC, their recovery ratios raised to 100 % for CA-PCL-DA-g-AH3 and CA-PCL-DA-g-AH6, and 97% for 29



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CA-PCL-DA-g-AH9, respectively. Moreover, CA-PCL-DA-g-AH3 can be deformed into complex shapes such as ‘eSMP’ (meaning ‘electroactive shape memory polyurethane’) (Figure 5f). These data demonstrated that all the as-prepared elastomers possessed shape memory capability, especially for CA-PCL elastomer, CA-PCL-DA0.3 elastomer and CA-PCL-DA-g-AH3 elastomer showing excellent shape recovery ratios and rapid recovery time at body temperature for biomedical applications. The shape memory property of the electroactive CA-PCL-DA-g-AH elastomer was further demonstrated by performing stress-controlled programming cycles test using DMA. Figure 5(g-i) showed one-way shape memory cycles of the three elastomers, and all of them presented good shape recovery ability after 5 cycles. Compared with CA-PCL elastomer and CA-PCL-DA0.3 elastomer, CA-PCL-DA-g-AH3 elastomer showed slightly less recovery ratio for its physical interactions from AH which caused chain motion. The results were consistent with the shape recovery results from water bath test (Figure 5e). For multiple cycles, there existed possible orientation of chains, thus the cyclic curves of all samples could not be completely overlapped. Furthermore, the temperature window for deformation recovery (Figure 5(g-i)) correlated well with the Tm of the elastomers (Figure 2f and Figure 5j) supporting that the entropy elasticity during the melting process was the driving force for shape recovery.

30


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Figure 5. Shape memory capacity evaluation of the elastomers in water baths (1: PCL80000;

2:

CA-PCL

elastomer;

3:

CA-PCL-DA0.3

elastomer;

4:

CA-PCL-DA-g-AH3; 5: CA-PCL-DA-g-AH6; 6: CA-PCL-DA-g-AH9) : (a) Initial shapes of the elastomers, (b) Fixed temporary shapes at 0 oC for 5 min, (c) The recovery shapes of the elastomers at 37 oC water bath, and (d) A shape memory cycle: fixed spiral shapes at 0 oC, spiral shapes peeled from the model, stable spiral shapes at 20 oC water bath and recovered initial shapes at 37 oC water bath. (e) Quantitative 31



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shape memory properties of the elastomers using water baths at 20 oC, 37 oC and 42 o

C, respectively, where Rf, Rr and T represented the shape fixity ratio, shape recovery

ratio and shape recovery time. (f) Character string of “eSMP” prepared by repeatedly using the same CA-PCL-DA-g-AH3 elastomer specimen: fix the letter form at 0 oC and recover the initial shape at 37 oC. One-way shape memory cycles (5 cycles) for CA-PCL elastomer (g), CA-PCL-DA0.3 elastomer (h) and CA-PCL-DA-g-AH3 elastomer (i). Starting with 0% tensile strain at 60 oC, all the specimens were subjected to consecutive cycles of tensile deformation (1), cooling (2), unloading of tensile stress (3), and recovering (4). (j) Storage modulus-temperature and loss angle (Tanẟ)-temperature curves. Scale bar: 1 cm. 3.8 Dynamic mechanical property The thermal mechanical properties of the CA-PCL elastomer, CA-PCL-DA0.3 elastomer and CA-PCL-DA-g-AH3 elastomer were further performed in this study. As shown in Figure 5j, all the CA-PCL elastomer, CA-PCL-DA0.3 elastomer and CA-PCL-DA-g-AH3 elastomer showed high modulus (crystalline modulus) from 79 MPa, 138 MPa to 187 MPa, respectively, when the temperature was below the fixation temperature. The storage modulus of the three specimens slowly decreased with the increase of temperature. However, when the temperatures were beyond their switching temperature and these samples were at the rubbery state, their storage modulus showed sharp decrease. The transition temperatures of the three films were 36 oC for CA-PCL-DA-g-AH3 elastomer, 34 oC for CA-PCL-DA0.3 elastomer and 31 o

C for CA-PCL elastomer, respectively, calculated from Figure 5j. The transition 32


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temperatures were close to the Tm from DSC curves in Figure S3. Moreover, the high elasticity ratios (the ratio of crystalline modulus to rubbery modulus)24 varied from 40, 64

to

93

folds

for

CA-PCL elastomer,

CA-PCL-DA0.3

elastomer

and

CA-PCL-DA-g-AH3 elastomer, and the high elasticity ratios endowed the materials with excellent shape fixity ratio as confirmed by Figure 5e. Besides, a large difference in modulus below and above the transition temperature and a sharp glass-rubber transition are the most substantial properties to render the materials shape memory function66. As shown in Figure 5j, CA-PCL elastomer and CA-PCL-DA0.3 elastomer with much lower transition temperatures of 31 oC and 34 oC, respectively, explained their higher recovery rate than that of CA-PCL-DA-g-AH3 elastomer with a higher transition temperature of 36 oC. 3.9 Cell viability and proliferation on the elastomers In skeletal muscle tissue engineering, one of the crucial parts is to establish a substrate that could facilitate the formation of functional myotubes67 which are the basic unit of the skeletal muscle tissue68. A material that could promote attachment, proliferation and differentiation of the myoblast may have positive effect on myogenesis. More importantly, materials used in tissue engineering should first have basic qualities such as

non-cytotoxicity

and

biocompatibility.

The

cytotoxicity

of

these

CA-PCL-DA-g-AH elastomers was assessed using PCL80000 film with good biocompatibility as a positive control. On the other hand, DA has been demonstrated to facilitate cell adhesion on the surface of materials69. DA in the elastomer may also have a positive effect on C2C12 adhesion and proliferation. Thus, CA-PCL elastomer 33



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film, possessing the same composition as CA-PCL-DA0.3 elastomer except for DA, was used as negative control. Compared with that on CA-PCL elastomer, cells on CA-PCL-DA0.3 elastomer showed a more normal morphology according to Live/Dead results in Figure 6a. C2C12 cells were spindle-shaped on the CA-PCL-DA0.3 elastomers while cells were almost in round shape on CA-PCL elastomers and PCL80000 films, suggesting that C2C12 cells attached and spread better on CA-PCL-DA0.3 elastomers than that on CA-PCL elastomers and PCL80000 films. Furthermore, from day 1 to day 3, the fluorescence intensity of the CA-PCL-DA0.3 elastomer group increased by about 2.2 folds, but that of the CA-PCL elastomer group only showed a 1.1 folds increase, indicating that DA also attributed to C2C12 cell proliferation (Figure 6b). Myoblasts are one kind of cells that could response to electrical stimuli and previous researches have demonstrated that the electroactive materials could promote myoblasts proliferation35, 40. In our study, the CA-PCL-DA-g-AH elastomers were endowed with electroactivity by the AH segment and were expected to promote the proliferation of C2C12 cells. From the Live/Dead assay results, the cell density on CA-PCL-DA-g-AH elastomers was higher than that on the CA-PCL-DA0.3 elastomers, but no obvious difference in the cell morphology could be observed, indicating that the addition of AH further promoted the cell attachment. AH, as an electroactive part in the CA-PCL-DA-g-AH elastomers, also showed a positive promotion on proliferation of C2C12 cells. Shown in Figure 6b, compared with non-AH groups (CA-PCL-DA0.3 elastomers), cells in AH contained groups 34


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(CA-PCL-DA-g-AH3, CA-PCL-DA-g-AH6 and CA-PCL-DA-g-AH9) exhibited higher fluorescence intensity on the same day (p

Dopamine-incorporated Dual Bioactive Electroactive Shape Memory

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