Synthesis, Characterization, and Electrospinning of a Functionalizable, Polycaprolactone-Based Polyurethane for Soft Tissue Engineering

polymers

Article Synthesis, Characterization, and Electrospinning of a Functionalizable, Polycaprolactone-Based Polyurethane for Soft Tissue Engineering

Jin-Jia Hu 1,*,† , Chia-Chi Liu 2,†, Chih-Hsun Lin 3,4,† and Ho-Yi Tuan-Mu 5,6

1 Department of Mechanical Engineering, National Yang Ming Chiao Tung University, Hsinchu 300, Taiwan 2 Department of Biomedical Engineering, National Cheng Kung University, Tainan 701, Taiwan; [email protected] 3 Division of Plastic and Reconstructive Surgery, Department of Surgery, Taipei Veterans General Hospital, Taipei 112, Taiwan; [email protected] 4 Department of Surgery, School of Medicine, National Yang Ming Chiao Tung University, Hsinchu 300, Taiwan 5 Department of Physical Therapy, Tzu Chi University, Hualien 970, Taiwan; [email protected] 6 Department of Sports Medicine Center, Hualien Tzu Chi Hospital, Buddhist Tzu Chi Medical Foundation, Hualien 970, Taiwan * Correspondence: [email protected]; Tel.: +886-3-5712121 (ext. 55110); Fax: +886-3-5720634 † These authors contributed equally to this work.

Abstract: We synthesized a biodegradable, elastomeric, and functionalizable polyurethane (PU) that can be electrospun for use as a scaffold in soft tissue engineering. The PU was synthesized from polycaprolactone diol, hexamethylene diisocyanate, and dimethylolpropionic acid (DMPA) chain extender using two-step polymerization and designated as PU-DMPA. A control PU using 1,4-butanediol (1,4-BDO) as a chain extender was synthesized similarly and designated as PU-BDO. Citation: Hu, J.-J.; Liu, C.-C.; Lin, The chemical structure of the two PUs was veriﬁed by FT-IR and 1H-NMR. The PU-DMPA had a C.-H.; Tuan-Mu, H.-Y. Synthesis, lower molecular weight than the PU-BDO (~16,700 Da vs. ~78,600 Da). The melting enthalpy of Characterization, and Electrospinning the PU-DMPA was greater than that of the PU-BDO. Both the PUs exhibited elastomeric behaviors of a Functionalizable, Polycaprolactone- with a comparable elongation at break (λ = L/L = 13.2). The PU-DMPA had a higher initial Based Polyurethane for Soft Tissue 0 Engineering. Polymers 2021, 13, 1527. modulus (19.8 MPa vs. 8.7 MPa) and a lower linear modulus (0.7 MPa vs. 1.2 MPa) and ultimate https://doi.org/10.3390/ strength (9.5 MPa vs. 13.8 MPa) than the PU-BDO. The PU-DMPA had better hydrophilicity than polym13091527 the PU-BDO. Both the PUs displayed no cytotoxicity, although the adhesion of human umbilical artery smooth muscle cells on the PU-DMPA surface was better. Bead free electrospun PU-DMPA Academic Editor: Zeynep Aytac membranes with a narrow ﬁber diameter distribution were successfully fabricated. As a demonstration of its functionalizability, gelatin was conjugated to the electrospun PU-DMPA membrane using Received: 8 April 2021 carbodiimide chemistry. Moreover, hyaluronic acid was immobilized on the amino-functionalized Accepted: 6 May 2021 PU-DMPA. In conclusion, the PU-DMPA has the potential to be used as a scaffold material for soft Published: 10 May 2021 tissue engineering.

Publisher’s Note: MDPI stays neutral Keywords: polyurethanes; two-step reaction; elastomer; functionalization; electrospinning; with regard to jurisdictional claims in 2,2-bis(hydroxymethyl)propionic acid published maps and institutional afﬁl- iations.

1. Introduction Soft tissue loss caused by trauma, post-tumor resection, aging, etc., can affect aesthetics Copyright: © 2021 by the authors. and even lead to exposure of vital structures of the body [1]. The replacement surgery Licensee MDPI, Basel, Switzerland. for treating massive soft tissue loss usually involves the use of autologous tissues, which This article is an open access article distributed under the terms and could cause donor site morbidity [2]. Tissue engineering integrating cells, biodegradable conditions of the Creative Commons scaffolds, and signaling may provide an alternative to autologous grafts for tissue repair Attribution (CC BY) license (https:// and regeneration [3]. In the process of tissue engineering, the biodegradable scaffold serves creativecommons.org/licenses/by/ as an initial support for cell adhesion and a guideline for microstructural development. 4.0/). Efforts have been made to synthesize polymers that can be used to fabricate scaffolds

Polymers 2021, 13, 1527. https://doi.org/10.3390/polym13091527 https://www.mdpi.com/journal/polymers Polymers 2021, 13, 1527 2 of 18

for repairing or regenerating various tissues. A polymer that is functionalizable and has mechanical properties comparable to the tissue to be replaced is more desired. Thermoplastic polyesters such as poly(glycolic acid) (PGA), poly(lactic acid) (PLA), polycaprolactone (PCL) and their copolymers have been widely used for tissue engineering applications due to their hydrolytic degradability [4]. The mechanical properties of PGA, PLA, PCL and their copolymers are not always adequate [5,6]. Crosslinked polyesters such as poly(glycerol sebacate) [7] and poly(1,8-octanediol citrate) [8] have several attractive features for use as scaffold materials. It is challenging to fabricate fibrous scaffolds from these crosslinked polyesters owing to their thermoset nature and insolubility [9–11]. Most polyesters tend to be less hydrophilic, which is unfavorable for cell adhesion [12]. In addition, chemical modification of the aforementioned polymers is not straightforward. Polyurethanes are a class of polymers composed of soft and hard segments joined by urethane links [13]. The soft segments are formed from mainly nonpolar polyols, whereas the hard segments from polar diisocyanates and chain extenders. The physiochemical and mechanical properties of polyurethanes are tunable by changing the composition of soft and hard segments. A variety of polyurethanes have been developed for repairing or regenerating soft tissues such as intervertebral discs, blood vessels and articular cartilages [14]. Scaffold materials may need certain functionality to meet their specific application requirements. For example, polymeric scaffolds to be used in contact with blood need to have proper blood compatibility, whereas hydrophilicity and antibacterial capability are important for those to be used as a wound dressing. Specific functionality can be provided if the polymer contains active functional groups such as carboxyl groups or amino groups. For example, Gong et al. prepared a polycarbonate-based polyurethane using dimethylolpropionic acid (DMPA), which contains a carboxyl group, as a chain extender. The carboxyl group-containing polyurethane was then modified by hyaluronan for better blood compatibility [15]. Notably, DMPA, either mixed with polyols or used as a chain extender, has been used to synthesize polyurethanes with tunable hydrophilicity [16], controllable degradability [17], reduced thrombogenicity [18], or stimuli sensitivity [19]. Polyurethanes have been fabricated into porous scaffolds by a variety of techniques [20–22]. Among them, electrospinning has received much attention as it re- quires a relatively simple setup and produces nonwoven meshes containing micro- and nanoscale fibers similar to many extracellular matrix components [23]. Aligned fibers that are capable of guiding the orientation of attached cells can potentially be used to control the neotissue microstructure [24]. The fiber formation during electrospinning is well explained by a theoretical model developed by Reneker and Yarin [25]. Electrospinning has been used in many other biomedical applications [26,27], as well as in fighting against the recent COVID-19 pandemic [28]. In this study, we aimed to develop a biodegradable, elastomeric, and functionalizable polyurethane for soft tissue engineering. PCL was selected as soft segments because of its hydrolyzability and low glass transition temperature (Tg). DMPA was used as a chain extender. Specifically, isocyanate-terminated PCL prepolymer was prepared in the first step and reacted with an equimolar amount of DMPA in the second step to form the polyurethane—that is, DMPA is the sole compound acting as the chain extender. A control polyurethane using 1,4-butanediol (1,4-BDO) as a chain extender was synthesized similarly under the same conditions. The chemical structure of the polyurethanes was confirmed by FTIR and 1H-NMR. Their mechanical tensile properties, hydrophilicity, and cytocompatibility were evaluated and compared. The polyurethane using DMPA as a chain extender was further electrospun to fabricate fibrous scaffolds. As a demonstration of the functionalizable carboxyl groups in the polyurethane, gelatin was incorporated to the scaffold by carbodiimide chemistry. Finally, we investigated the possibility of conjugation of hyaluronan on the surface of the functionalizable polyurethane. Polymers 2021, 13, 1527 3 of 18

2. Materials and Methods 2.1. Materials Polycaprolactone diol (PCL diol; PLACCEL 220, Mw = 2000) was obtained from Daicel (Japan). Hexamethylene diisocyanate (HDI, 98.5%) was obtained from Fluka (Switzerland). 1,4-Butanediol (1,4-BDO, 99%) was obtained from Acros (Morris Plains, NJ, USA). Dimethyl acetamide (DMAc, 99.9%) was obtained from Tedia (Fairfield, OH, USA). Stannous octoate 0 (Sn(Oct)2), dimethyl sulfoxide-d6, N-(3-Dimethylaminopropyl)-N -ethylcarbodiimide hy- drochloride (EDC), gelatin from porcine skin, Ponceau S solution, and hyaluronan sodium salt (HA) were obtained from Sigma-Aldrich (St Louis, MO, USA). Dibutylamine was obtained from J.T. Baker (Phillipsburg, NJ, USA). 1,1,1,3,3,3-Hexafluoro-2-propanol (99%) was obtained from Matrix Scientific (Columbia, SC, USA). 2,2-Bis(hydroxymethyl)propionic acid (DMPA), 1,4-diaminobutane (1,4-BDA, 98.5%) and N-hydroxysuccinimide (NHS) were obtained from Alfa Aesar (Lancashire, UK). Tetrahydrofuran (THF) was purchased from Macron (Center Valley, PA, USA). Poly-L-lysine (MW = 12,000 Da) was kindly provided by Prof. Jeng-Shiung Jan. PCL diol was dried at 120 ◦C under vacuum for 4 h prior to use. 1,4-BDO and HDI were purified by distillation and stored over molecular sieves (4 Å, Sigma-Aldrich). DMAc was dried over molecular sieves for more than two days. The water content of PCL diol, HDI, 1,4-BDO, and DMAc was determined by a Karl Fischer titrator (831 Coulometer, Metrohm, Switzerland) to ensure that the water content is less than 100 ppm. DMPA and all other reagents were used as received. For cell culture, Dul- becco’s modified Eagle’s medium (DMEM), penicillin/streptomycin, and trypsin-EDTA were obtained from Thermo Fisher Scientific (Waltham, MA, USA) and fetal bovine serum was purchased from Hyclone (Logan, UT, USA).

2.2. Synthesis of Polyurethanes In this study, polyurethanes were synthesized by two-step solution polymerization (Scheme1). The synthesis was performed under a dry nitrogen atmosphere in a 250 mL four-necked reactor equipped with a mechanical stirrer. The stoichiometry of the reaction was 2:1:1 of HDI: PCL diol: chain extender (DMPA or 1,4-BDO). In the ﬁrst step, isocyanate- terminated PCL prepolymer was prepared. A 50% (w/v) solution of PCL diol in DMAc was heated to 65 ◦C in the reactor with continuous stirring. HDI and a catalytic amount of Sn(Oct)2 were then added. The prepolymerization reaction was allowed to continue at 65 ◦C until the isocyanate content reached the theoretical value determined by dibutylamine titration. Subsequently, in the second step, a solution of DMPA or 1,4-BDO was added to the prepolymer solution, initiating chain extension. The reaction was continued until the NCO peak at 2270 cm−1 in the Fourier transform infrared (FT-IR) spectrum of the reaction mixture was not detected. The product was precipitated in distilled water, ﬁltered, and dried under vacuum at 60 ◦C for 24 h. The polymer was designated as PU-DMPA or PU-BDO according to the chain extender used. Polymers 2021, 13, 1527 4 of 18 Polymers 2021, 13, x FOR PEER REVIEW 4 of 20

SchemeScheme 1.1. Two-step synthesis synthesis of of polyurethanes: polyurethanes: (a) synthesis (a) synthesis of isocyanate-terminated of isocyanate-terminated prepoly- prepoly- mer;mer; ((bb)) reactionreaction of the the prepolymer prepolymer with with a adiol diol chai chainn extender extender to establish to establish high-molecular-weight high-molecular-weight polyurethane.polyurethane.

2.3.2.3. CastCast FilmFilm Preparation TheThe PU-DMPAPU-DMPA and the the PU-BDO PU-BDO were were dissolved dissolved in inHFIP HFIP and and DMF, DMF, respectively, respectively, to to prepareprepare 5%5% ((w/vw/v) solutions. The The solution solution was was then then poured poured into into a Teflon a Teflon dish dish (7.5 (7.5 cm cmin in diameter)diameter) andand degasseddegassed underunder vacuumvacuum atat roomroom temperature.temperature. Upon the removal of bubbles,bub- thebles, PU-DMPA the PU-DMPA and the and PU-BDO the PU-BDO solutions solutions were were dried dried in a fumein a fume hood hood at room at room temperature tem- andperature under and vacuum under atvacuum 60 ◦C, at respectively, 60 °C, respective for 24ly, h, for to form24 h, films.to form The films. thickness The thickness of the film wasof the measured film was bymeasured a custom by high-frequency a custom high-frequency ultrasound ultrasound system. system.

2.4.2.4. MaterialMaterial Characterization 2.4.1.2.4.1. GelGel PermeationPermeation Chromatography Chromatography TheThe molecular weights weights of of the the PU-DMPA PU-DMPA and and the thePU-BDO PU-BDO were weredetermined determined by a gel by a gelpermeation permeation chromatography chromatography and and light-scatte light-scatteringring (GPC–LS) (GPC–LS) system system (Viscotek, (Viscotek, United United Kingdom)Kingdom) equippedequipped with with two two Shodex Shodex HFIP HFIP columns columns and and three three detectors detectors including including RI RI (VE3580,(VE3580, Viscotek),Viscotek), right angle angle light light scatteri scattering,ng, and and viscometer viscometer (Dual (Dual 270, 270, Viscotek). Viscotek). HFIP HFIP atata a ﬂowflow raterate ofof 1.0 mL/min was was used used as as the the eluent. eluent.

2.4.2. FT-IR Spectroscopy The chemical structure of the PU-DMPA and the PU-BDO was examined by infrared spectroscopy. FT-IR spectra of samples were obtained by scanning the samples for 64 times in the wave number range from 600 cm−1 to 4000 cm−1 at the resolution of 2 cm−1 using a

Polymers 2021, 13, 1527 5 of 18

Nicolet 6700 FT-IR spectrometer (Thermo Fisher Scientiﬁc, Waltham, MA, USA) with an ATR module.

2.4.3. Proton Nuclear Magnetic Resonance Samples of the PU-DMPA and the PU-BDO were dissolved in dimethyl sulfoxide- 1 d6 to prepare 20 mg/mL solutions. H NMR spectra (500 MHz) were recorded on an Avance 500 NMR spectrometer (Bruker, Germany). Chemical shifts were given in ppm with tetramethylsilane as the standard.

2.4.4. Differential Scanning Calorimetry The melting behavior and crystallization of the PU-DMPA and the PU-BDO were investigated by a Perkin-Elmer DSC7 (Perkin-Elmer, Norwalk, CT, USA). Samples weighted 6 mg were examined. The sample chamber was purged with nitrogen gas at a ﬂow rate of 50 mL/h. The temperature sequence was arranged as follows: the sample was heated to 280 ◦C at 10 ◦C/min, equilibrated at 280 ◦C, and cooled to −40 ◦C at 10 ◦C/min. Then the sample was heated again to 280 ◦C at 10 ◦C/min.

2.4.5. Mechanical Tensile Testing The mechanical behavior of the PU-DMPA and the PU-BDO was estimated by uniaxial mechanical tensile testing. Dog-bone shaped specimens were punched from the PU-DMPA and the PU-BDO cast ﬁlms using a miniature ASTM D412-C die (gauge length: 16.5 mm; width: 3 mm). The specimen was mounted on an ATS machine (AG-1, Shimadzu, Japan) equipped with a 200 N load cell (Shimadzu, Japan). A tensile test was conducted on under a crosshead speed of 30 mm/min until membrane break. The force (N) and displacement (mm) were recorded. The engineering stress was calculated as force divided by original cross-sectional area and the stretch ratio was calculated as (displacement + 16.5)/16.5. The stress–stretch curves of the specimen were plotted and the ultimate stress, elongation at break and initial modulus of the specimen were obtained.

2.4.6. Water Contact Angle Measurements The wettability of the PU-DMPA and the PU-BDO cast ﬁlms was assessed by water contact angle measurements. Water drops of 8 µL were generated by a micropipette and carefully deposited on the surface of the specimens. Photographs of the droplets on the surface were acquired by a digital camera and the contact angles were measured by ImageJ (NIH) with an average of six measurements for each sample.

2.5. Cell Adhesion Tests Human umbilical artery smooth muscle cells (HUASMCs) were used to investigate cell adhesion behavior on the surface of the PU-DMPA and the PU-BDO cast films. HUASMCs were isolated according to an established protocol [9] and cultured in DMEM supplemented ◦ by 10% fetal bovine serum and 1% penicillin/streptomycin at 37 C in a CO2 incubator. Before reaching confluence, cells were detached by 0.05% trypsin-EDTA, counted, and resuspended in culture medium for experiments. The PU-DMPA and the PU-BDO cast films were sterilized by immersion in 70% ethanol for 30 min, followed by washing with PBS and then exposure to UV irradiation in a laminar flow hood overnight. The HUASMCs were then seeded on the top of the film or in tissue culture polystyrene dishes at a density of 2.5 × 104/cm2. After 24 h, the cell seeded film was washed with PBS and then fixed by 4% paraformaldehyde for 30 min. Images of the attached cells on the film were acquired with an inverted optical microscope (DM IL LED, Leica, Germany) equipped with a digital camera (EOS 7000, Canon, Japan).

2.6. Immobilization of Gelatin The PU-DMPA cast ﬁlm (2 cm × 2 cm, ~50 mg) was placed in a solution of EDC (0.2 g) and NHS (0.05 g) in MES buffer (0.5 M, pH = 5.5) (10 mL) for 30 min to activate carboxyl Polymers 2021, 13, x FOR PEER REVIEW 6 of 20

film were acquired with an inverted optical microscope (DM IL LED, Leica, Germany) equipped with a digital camera (EOS 7000, Canon, Japan).

Polymers 2021, 13, 1527 2.6. Immobilization of Gelatin 6 of 18 The PU-DMPA cast film (2 cm × 2 cm, ~50 mg) was placed in a solution of EDC (0.2 g) and NHS (0.05 g) in MES buffer (0.5 M, pH = 5.5) (10 mL) for 30 min to activate carboxyl groups on the film surface. After a brief water in MES buffer, the film was transferred to groups on the film surface. After a brief water in MES buffer, the film was transferred a 10% (w/v) gelatin in PBS solution (10 mL) to allow the activated carboxyl groups to react to a 10% (w/v) gelatin in PBS solution (10 mL) to allow the activated carboxyl groups to reactwith the with amino the amino groups groups in gelatin in gelatin for 4 h forat 40 4 h°C at (Scheme 40 ◦C (Scheme 2). The2 result). The of result gelatin of grafting gelatin graftingwas verified was verifiedby FT-IR by and FT-IR Ponceau and Ponceau S staini Sng; staining; the latter the latter was wasperformed performed following following the thestandard standard protocol. protocol.

Scheme 2. Grafting of primary amines to the PU-DMPA backbone upon carbodiimide-mediated Scheme 2. Grafting of primary amines to the PU-DMPA backbone upon carbodiimide-mediated carboxyl activation. carboxyl activation. 2.7. Cytotoxicity Tests 2.7. Cytotoxicity Tests The cytotoxicity of the PU-DMPA, gelatin-modified PU-DMPA, and PU-BDO cast filmsThe was cytotoxicity examined byof athe direct PU-DMPA, contact gelatin-modified method. HUASMCs PU-DMPA, were seeded and PU-BDO in a 48-well cast platefilms was at 2.5 examined× 104 cells by a per direct well. contact Upon meth cellod. adhesion, HUASMCs a piece were of seeded 2.5 mg in of a the 48-well sterilized plate PU-DMPA,at 2.5 × 104 cells gelatin-modified per well. Upon PU-DMPA, cell adhesion, or PU-BDOa piece of cast2.5 mg film of was the sterilized placed on PU-DMPA, top of the cells.gelatin-modified The metabolic PU-DMPA, activity of or the PU-BDO cells was cast assessed film was by placed Alamar on Blue top of assay the oncells. days The 2, met- 4, 6 andabolic 8 usingactivity the of following the cells equationwas assessed [29]: by Alamar Blue assay on days 2, 4, 6 and 8 using the following equation [29]: A − (A × R ) ( ) = 562 −×620 o × Percentage of reduced Alamar Blue % AAR562() 620 o 100 Percentage of reduced Alamar Blue (%) =A562 × 100 A562 where A562 and A620 are the absorbance of test wells at 562 nm and 620 nm, respectively; Ro A A iswhere the correction 562 andfactor 620 for are filtersthe absorbance =Ao,562/A ofo,620 test; Awellso,562 atis 562 the absorbancenm and 620 atnm, 562 respectively; nm due to

oxidizedRo is the Alamar correction Blue; factor and Aforo,620 filtersis the = absorbanceAAoo,562 ,620 ; atA 620o,562 nm, is the due absorbance to the same at oxidized 562 nm Ala-due mar Blue. The absorbance was quantified by a microplate reader (Multiskan FC; Waltham, to oxidized Alamar Blue; and Ao,620 is the absorbance at 620 nm, due to the same oxidized MA, USA, Thermo Scientific). At least five specimens for each group were analyzed.

2.8. Electrospinning PU-DMPA ﬁbrous membranes were prepared by electrospinning using a custom setup which consists of a spinneret (21-gage needle), a syringe pump (KDS-210, KD Scientiﬁc, Holliston, MA), a high-voltage DC power supply (AU series, Matsusada Precision, Japan), and a grounded aluminum rotating drum (102 mm in diameter) driven by a high-speed Polymers 2021, 13, 1527 7 of 18

DC motor [9]. Briefly, the PU-DMPA was dissolved in HFIP to prepare 15% (w/v) solution. The solution was delivered by the syringe pump at a flow rate of 1.5 mL/h to the spinneret, on which a positive charge of ~10 kV was applied, and electrospun onto the rotating drum. A 25 cm tip-to-drum distance was employed. Fibers were collected for 2 h to form a membrane. The thickness of the membranes, measured by the high-frequency ultrasound system, ranged between 170 and 220 µm. Immobilization of gelatin on the electrospun PU-DMPA membrane was performed following the procedure described earlier. The result was also verified by FT-IR and Ponceau S staining. The porosity of the PU-DMPA electrospun membranes, before and after gelatin immobilization, was measured by a water displacement method [29]. Briefly, a piece of the membranes was immersed in a 5 mL graduated cylinder filled with a known volume of water, V1. The increased volume, V2 − V1, representing the volume of solid material, Vsolid, was measured upon the removal of the air in the pores of the membrane. The volume of the water in the cylinder, V3, was measured again after the water-impregnated membrane was removed. The difference between V3 and V1 was identified as the void volume, Vvoid. The porosity was calculated as φ = (V1 − V3)/(V2 − V3).

2.9. Scanning Electron Microscopy The morphology of electrospun PU-DMPA fibers before and after gelatin immobilization was examined by scanning electron microscopy (SEM). Pieces of the polyurethane membranes (3 mm × 5 mm) were mounted onto stubs and sputter-coated with platinum (Sputter E-1045, Hitachi, Japan). SEM images were taken by a field emission scanning electron microscope (S-4100, Hitachi, Japan). The voltage used was 15 kV. The vacuum was 5 × 10−8 Pa. The diameters of electrospun PU-DMPA fibers, before and after gelatin immobilization, were measured using ImageJ with an average of 30 measurements from three SEM images of each polyurethane.

2.10. Amino Functionalization and Modification by Hyaluronic Acid Based on Scheme2, the PU-DMPA was amino-functionalized by two approaches. In one approach, 1,4-BDA was grafted directly onto the PU-DMPA cast film following the procedure described earlier; the resultant membrane is designated as PU-DMPA-BDA. In the other approach, the PU-DMPA (1 g) was dissolved in THF (50 mL) to prepare a 2% (w/v) solution. An excess amount of EDC (0.72 g) and NHS (0.18 g) in methanol (12 mL) was gradually added to the PU-DMPA solution. After reaction for 30 min, a solution of poly-L-lysine (0.06 g) in methanol (1.5 mL) was added to the solution. The reaction continued for 24 h with agitation. The solution was then casted in a Teflon dish and dried at room temperature to obtain a membrane, designated as PU-DMPA-PLL. The result of amino functionalization was verified by FT-IR and Ponceau S staining. For immobilization of hyaluronic acid (HA) on the amino-functionalized PU-DMPA, EDC and NHS were added to 0.5% (w/v) HA in MES buffer; the final concentrations of EDC and NHS were 6% (w/v) and 1.5% (w/v), respectively. The amino-functionalized PU-DMPA film (2 cm × 2 cm, ~50 mg), either PU-DMPA-BDA or PU-DMPA-PLL, were added to the HA buffer (10 mL) with stirring at 40 ◦C for 24 h.

2.11. Platelet Adhesion Testing Human whole blood was collected from healthy human donors. Anticoagulated blood (blood/anticoagulant ratio of 9:1 (v/v)) was centrifuged (Model 2420, Kubota, Japan) at 1500 rpm for 15 min to obtain plasma. The PU-DMPA, PU-DMPA-BDA, PU-DMPA-PLL, PU-DMPA-BDA-HA and PU-DMPA-PLL-HA membranes with a size of 1 cm × 1 cm were incubated in the plasma at 37 ◦C for 1 h. The membranes were then washed with PBS to remove unattached platelets and ﬁxed with a 2.5% glutaraldehyde solution at 4 ◦C for 2 h. Upon drying through graded alcohols, the membranes were sputter-coated with platinum and the morphology of platelet on the surfaces of the membranes was examined by scanning electron microscopy. Polymers 2021, 13, 1527 8 of 18

2.12. Statistical Analysis All the data are presented as mean ± standard deviation. The Student’s t test was used to compare the mechanical properties and the contact angles of the PU-DMPA and the PU-BDO. One-way ANOVA in conjunction with Holm-Sodal post hoc procedure was performed to examine the difference in cytotoxicity. The level of signiﬁcance was set at 0.05. A * denotes p < 0.05, while ** denotes p < 0.001.

3. Results and Discussion The goal of this study is to synthesize an elastomeric polyurethane that can be electrospun and contains carboxyl groups for functionalization. The PU-DMAP and the PU-BDO were synthesized by two-step polymerization under the same conditions. The time required to complete the chain extension of the PU-DMPA and the PU-BDO was 4 days and 24 h, respectively, which indicates the very different reaction activity between DMPA and 1,4-BDO as a chain extender. The molecular weights and molecular weight distributions of the polyurethanes were obtained from GPC results (see Supplementary Figure S1 for GPC chromatograms). The molecular weight of the PU-DMPA (Mw = ~16,700 Da, PDI = 1.31) was signiﬁcantly lower than that of the PU-BDO (Mw = ~78,600 Da, PDI = 1.60), probably because the hydroxyl groups of DMPA reacted less readily with isocyanate-terminated PCL prepolymer owing to the steric hindrance. Figure1 shows the FT-IR spectra of the PU-DMPA and the PU-BDO. The absence of peaks around 2270 cm−1, which is a characteristic absorption for N=C stretching, indicates that isocyanates were completely reacted. The emergence of peaks around 1530 cm−1, 1700 cm−1, and 3300 cm−1, which were assigned to the vibrations of N-H bending, C=O Polymers 2021, 13, x FOR PEER REVIEWstretching, and N-H stretching of the urethane group, respectively, demonstrates9 theof 20

formation of the urethane linkage.

FigureFigure 1. 1.ATR-FTIR ATR-FTIR spectra spectra of of the the PCL-diol PCL-diol ( a(a),), the the PU-BDO PU-BDO ( b(b)) and and the the PU-DMPA PU-DMPA (c ().c).

Generally,Generally, C=O C=O stretching stretching vibration vibration of of esters esters and and urethanes urethanes manifests manifests itself itself as as peaks peaks between 1600 cm−1 and 1800 cm−1. In the PU-BDO spectrum, the peak at 1735 cm−1 and between 1600 cm−1 and 1800 cm−1. In the PU-BDO spectrum, the peak at 1735 cm−1 and the the shoulder peak at 1685 cm−1 were assigned to free ester or urethane carbonyl groups and shoulder peak at 1685 cm−1 were assigned to free ester or urethane carbonyl groups and hydrogen-bonded (H-bonded) ester or urethane carbonyl groups, respectively [30,31]. In hydrogen-bonded (H-bonded) ester or urethane carbonyl groups, respectively [30,31]. In the PU-DMPA spectrum, although no separate peaks were observed in this region, the peak the PU-DMPA spectrum, although no separate peaks were observed in this region, the at 1726 cm−1 was strong and broad compared to the two peaks for the PU-BDO. Note that, peak at 1726 cm−1 was strong and broad compared to the two peaks for the PU-BDO. Note in addition to the carboxyl group in soft segments and urethane groups in hard segments, that, in addition to the carboxyl group in soft segments and urethane groups in hard seg- there are carboxyl groups in the hard segments and all these groups might contribute to ments, there are carboxyl groups in the hard segments and all these groups might contribute to H-bonded carbonyl groups. Both the PU-BDO and the PU-DMPA exhibited 2936 cm−1 and 2861 cm−1 absorptions, corresponding to symmetric and asymmetric C-H stretching from CH2, respectively. The peaks around 3300 cm−1 were assigned to N-H stretching of urethane amide (for both the PU-DMPA and the PU-BDO) or O-H stretching in the carboxyl group (for the PU-DMPA only). The shifting of the peaks to the low wave number for both the PU- DMPA and the PU-BDO also indicates the formation of hydrogen bonding. Figure 2 shows the 1H-NMR spectra of the PU-DMPA and the PU-BDO with each peak assigned. The PU-DMPA and the PU-BDO spectra shared many common peaks, including: 1.2 ppm (peak a), 1.3 ppm (peak b), 1.5 ppm (peaks c-e), 2.25 ppm (peak f), 2.95 ppm (peak g), 3.6 ppm (peak h), 3.98 ppm (peak i), and 4.12 ppm (peak j). These peaks were assigned to proton signals of HDI and PCL units. Peaks d’ and j’ in the PU-BDO spectrum were assigned to proton signals of 1,4-BDO units, whereas peaks a’ and i’ in the PU-DMPA spectrum were DMPA, indicating the chain extenders were incorporated into the polymers, respectively. The results of 1H-NMR were consistent with the findings in the FT-IR; both support that both the polyurethanes were synthesized as designed. Figure 3 shows DSC curves of the PU-DMPA and the PU-BDO. The glass transition temperature (Tg) of both the polyurethanes were below room temperature, which indicates that both the polyurethanes are in a rubbery state at room temperature or under physiological conditions. The PU-DMPA had a slightly lower Tg (2.5 °C) compared to the PU-BDO (6.8 °C). Both the PU-DMPA and the PU-BDO displayed melting peaks, which is due to the crystallinity of PCL segments. Similarly, the melting temperature (Tm) of the PU-DMPA (39.2 °C) was slightly lower than that of the PU-BDO (41.3 °C). The reduction in Tg and Tm might be attributed to the compromised crystallinity of PCL soft segments

Polymers 2021, 13, 1527 9 of 18

H-bonded carbonyl groups. Both the PU-BDO and the PU-DMPA exhibited 2936 cm−1 and 2861 cm−1 absorptions, corresponding to symmetric and asymmetric C-H stretching from CH2, respectively. The peaks around 3300 cm−1 were assigned to N-H stretching of urethane amide (for Polymers 2021, 13, x FOR PEER REVIEWboth the PU-DMPA and the PU-BDO) or O-H stretching in the carboxyl group (for10 of the 20

PU-DMPA only). The shifting of the peaks to the low wave number for both the PU-DMPA and the PU-BDO also indicates the formation of hydrogen bonding. 1 causedFigure by hydrogen2 shows thebondsH-NMR between spectra the hard of segments the PU-DMPA (N-H andin urethane the PU-BDO linkage with and each ad- ditionalpeak assigned. O-H owing The to PU-DMPA the carboxyl and group the PU-BDOin DMPA) spectra and the shared soft segments many common (PCL carbonyl peaks, groups)including: [32]. 1.2 Additionally, ppm (peak a),the 1.3 asymmetric ppm (peak side b), 1.5chains ppm of (peaks DMPA c-e), in hard 2.25 ppmsegments, (peak in- f), cluding2.95 ppm a (peakmethyl g), group 3.6 ppm and (peak a carboxyl h), 3.98 group, ppm (peakmight i), impede and 4.12 an ppm ordered (peak packing j). These of peaks hard segments,were assigned which to can proton act as signals a physical of HDI crosslinker. and PCL units.Interestingly, Peaksd’ the and melting j’ in the enthalpy PU-BDO of spectrum were assigned to proton signals of 1,4-BDO units, whereas peaks a’ and i’ in the the PU-DMPA was significantly greater than that of the PU-BDO, which might be ex- PU-DMPA spectrum were DMPA, indicating the chain extenders were incorporated into plained by the additional hydrogen bonding introduced by the carboxyl groups of the PU- the polymers, respectively. The results of 1H-NMR were consistent with the ﬁndings in the DMPA. FT-IR; both support that both the polyurethanes were synthesized as designed.

1 Figure 2. 1H-NMRH-NMR of of the the PU-BDO PU-BDO ( (toptop)) and and the the PU-DMPA PU-DMPA ( (bottom).).

Polymers 2021, 13, 1527 10 of 18

Figure3 shows DSC curves of the PU-DMPA and the PU-BDO. The glass transition temperature (Tg) of both the polyurethanes were below room temperature, which indicates that both the polyurethanes are in a rubbery state at room temperature or under physiolog- ◦ ical conditions. The PU-DMPA had a slightly lower Tg (2.5 C) compared to the PU-BDO (6.8 ◦C). Both the PU-DMPA and the PU-BDO displayed melting peaks, which is due to the crystallinity of PCL segments. Similarly, the melting temperature (Tm) of the PU-DMPA ◦ ◦ (39.2 C) was slightly lower than that of the PU-BDO (41.3 C). The reduction in Tg and Tm might be attributed to the compromised crystallinity of PCL soft segments caused by hydrogen bonds between the hard segments (N-H in urethane linkage and additional O-H owing to the carboxyl group in DMPA) and the soft segments (PCL carbonyl groups) [32]. Additionally, the asymmetric side chains of DMPA in hard segments, including a methyl group and a carboxyl group, might impede an ordered packing of hard segments, which can act as a physical crosslinker. Interestingly, the melting enthalpy of the PU-DMPA was Polymers 2021, 13, x FOR PEER REVIEW 11 of 20 signiﬁcantly greater than that of the PU-BDO, which might be explained by the additional hydrogen bonding introduced by the carboxyl groups of the PU-DMPA.

FigureFigure 3. 3.DSC DSC crystallization crystallization curves curves (left (left) and) and second second melting melting curves curves (right (right) of) of the the PU-BDO PU-BDO (a), (a and), theand PU-DMPA the PU-DMPA (b). (b).

FigureFigure4 shows4 shows the the average average stress–stretch stress–stretch curves curves of of the the PU-DMPA PU-DMPA and and the the PU-BDO PU-BDO andand the the comparisons comparisons of of their their mechanical mechanical properties. properties. Both Both the the polyurethanes polyurethanes exhibited exhibited λ == elastomericelastomeric behavior behavior with with a comparablea comparable elongation elongation at breakat break (λ =( L/LLL0 0=13.2).13.2). TheThe PU- PU- DMPADMPA had had a a higher higher initial initial modulus modulus (19.8 (19.8 MPa MPa vs. vs. 8.7 8.7 MPa) MPa) and and a a lower lower linear linear modulus modulus (0.7(0.7 MPa MPa vs. vs. 1.2 1.2 MPa) MPa) and and ultimate ultimate strength strength (9.5 (9.5 MPa MPa vs. vs. 13.8 13.8 MPa) MPa) than than the the PU-BDO. PU-BDO. NoteNote that that the the mechanical mechanical properties properties of of the the PU-BDO PU-BDO are are comparable comparable to to those those reported reported by by HongHong et et al. al. [18 [18].]. The The elasticity elasticity of of polyurethanes polyurethanes is is derived derived from from the the phase phase separation separation of of thethe soft soft and and hard hard segments segments in in a a way way that that the the hard hard segment segment domains domains serve serve as as crosslinks crosslinks amongamong the the relativelyrelatively amorphousamorphous soft segment segment domains. domains. The The side side chains chains of DMPA, of DMPA, in con- in contrasttrast to the to the linear linear 1,4-BDO, 1,4-BDO, might might impede impede the packing the packing of hard of hardsegments segments of the of PU-DMPA the PU- DMPAand hence and hencecontribute contribute to the to phase the phase mixing mixing between between soft softand andhard hard segments, segments, leading leading to a tocompromised a compromised linear linear modulus modulus and and ultimate ultimate strength strength [33]. [33 ].The The greater greater initial initial modulus modulus of ofthe the PU-DMPA, PU-DMPA, which which was was estimated estimated before before the the stretch stretch ratio ratio reached reached 1.2, 1.2, might might be be at- attributedtributed to to the the additional additional hydrogen hydrogen bonding bonding introduced introduced by the by carboxyl the carboxyl groups groups of the PU- of theDMPA. PU-DMPA. Recently, Recently, Mamidi Mamidi et al. fabricated et al. fabricated carbon nano-onions carbon nano-onions reinforced reinforced PCL composite PCL compositenanofibers nanofibers for pH-responsive for pH-responsive drug release drug [34]. release The incorporation [34]. The incorporation of carbon nano-onions of carbon nano-onionsenhanced the enhanced tensile properties the tensile propertiesof PCL nano offibers. PCL nanofibers. When compared Whencompared with their with composite their compositenanofibers, nanofibers, the PU-DMPA the PU-DMPA cast film castwas filmstronger was strongerand more and flexible, more flexible,probably probably owing to owingthe characteristic to the characteristic interactions interactions between between soft and soft hard and segments. hard segments. The modulus The modulus of the PU- of the PU-DMPA was comparable to some biological soft tissues, such as skin [35] and blood DMPA was comparable to some biological soft tissues, such as skin [35] and blood vessels vessels [36], and it is thus particularly suitable to be used in these areas. [36], and it is thus particularly suitable to be used in these areas.

Polymers 2021, 13, 1527 11 of 18 Polymers 2021, 13, x FOR PEER REVIEW 12 of 20

Figure 4. Average stress–stretch curves and mechanical properties of the PU-DMPA and the PU-BDO. Figure* 4. denotes Averagep < stress–stretch 0.05, while ** curves denotes andp < mechanic 0.001. al properties of the PU-DMPA and the PU- BDO. * denotes p < 0.05, while ** denotes p < 0.001. Scaffold materials for tissue engineering should have proper surface hydrophilicity Scaffoldfor cells materials to adhere for to. Atissue moderate engineering surface should wettability have (50–70 proper◦) hassurface been hydrophilicity shown to favor cell for cellsadhesion to adhere and to. growth A moderate [37]. Figure surface5a wettability shows that (50–70°) the water has contact been shown angle ofto thefavor PU-DMPA cell adhesion(~60 and◦) was growth significantly [37]. Figure smaller 5a shows than that that the of water the PU-BDO contact (~75angle◦) of and the in PU-DMPA the range that (~60°)supports was significantly cell adhesion smaller [37 than]. The that improved of the PU-BDO wettability (~75°) might and in be the due range to the that presence sup- of ports carboxylcell adhesion groups [37]. in The the PU-DMPAimproved wettability backbone, implyingmight be thatdue theto the carboxyl presence group of ofcar- DMPA boxyl remainsgroups in intact the PU-DMPA upon reaction. backbone, The result implyi is consistentng that the with carboxyl Chen et group al. [16 of]. FigureDMPA5 bre- shows mainsthe intact morphology upon reaction. of HUASMCs The result on is the consis surfacetent ofwith the Chen PU-DMPA et al. [16]. and theFigure PU-BDO 5b shows cast films. the morphologyThe cells adhered of HUASMCs and spread on the on surfac the PU-DMPAe of the PU-DMPA surface. Particularly, and the PU-BDO most of cast the cells films. displayedThe cells adhered lamellipodia, and spread which suggestson the PU-DMPA that they weresurface. migrating. Particularly, The morphology most of the of the cells displayedcells on the lamellipodia, PU-BDO surface which was suggests quite different: that they most were of migrating. the cells were The round morphology in shape and of theonly cells aon few the spread PU-BDO out—that surface is, was the PU-DMPAquite different: had bettermost of cell the adhesion cells were than round the PU-BDO, in shapewhich and only might a few be spread due to theout—that better hydrophilicityis, the PU-DMPA of thehad PU-DMPA. better cell adhesion than the PU-BDO, whichAfter gelatinmight be modification, due to the be thetterwater hydrophilicity contact angle of the of PU-DMPA. the PU-DMPA films slightly decreased to 57 ± 2.8◦. The reduction in contact angle was not significant, however. Figure6 shows that there were no differences in the metabolic activity of HUASMCs cultured with the PU-DMPA, gelatin-modified PU-DMPA and PU-BDO cast films and those grown alone in tissue culture plates throughout the eight-day culture, indicating their excellent cytocompatibility.

Polymers 2021, 13, x FOR PEER REVIEW 13 of 20

Polymers 2021, 13, 1527 12 of 18 Polymers 2021, 13, x FOR PEER REVIEW 13 of 20

Figure 5. (a) Representative photographs of water droplets on the surface of the PU-BDO and the PU-DMPA cast films and the comparison of their contact angles. (b) Morphology of HUASMCs adhered on the surface of the PU-BDO and the PU-DMPA cast films after one day of culture; scale bar = 100 µm. * denotes p < 0.05.

After gelatin modification, the water contact angle of the PU-DMPA films slightly decreased to 57 ± 2.8°. The reduction in contact angle was not significant, however. Figure Figure6 showsFigure 5. (athat) 5. Representative( athere) Representative were photographsno differences photographs of water in of the water drople metabolic dropletsts on the activity on surface the surface of of HUASMCsthe of PU-BDO the PU-BDO andcultured the and the PU-DMPAwithPU-DMPA the PU-DMPA, cast castfilms ﬁlms and gelatin-modified the and compar the comparisonison ofPU-DMP their of contact theirA and contact angles. PU-BDO angles. (b) Morphology cast (b) films Morphology and of HUASMCs those of HUASMCsgrown adheredaloneadhered in on tissue the on surface theculture surface of platesthe of PU-BDO the throughout PU-BDO and the and the PU-D the eight-day PU-DMPAMPA cast culture, films cast ﬁlms after indicating afterone day one of daytheir culture; of excellent culture; scale scale barcytocompatibility. =bar 100 = µm. 100 µ* m.denotes * denotes p < 0.05.p < 0.05.

After gelatin modification, the water contact angle of the PU-DMPA films slightly decreased to 57 ± 2.8°. The reduction in contact angle was not significant, however. Figure 6 shows that there were no differences in the metabolic activity of HUASMCs cultured with the PU-DMPA, gelatin-modified PU-DMPA and PU-BDO cast films and those grown alone in tissue culture plates throughout the eight-day culture, indicating their excellent cytocompatibility.

FigureFigure 6. Growth 6. Growth of HUASMCs of HUASMCs cultured cultured alone alone in ti inssue tissue culture culture plates, plates, with with the the PU-BDO, PU-BDO, PU- PU-DMPA, DMPA,and and gelatin-modified gelatin-modified PU-DMPA, PU-DMPA, respectively, respectively in terms, in terms of the of percentage the percentage of reduced of reduced Alamar Ala- blue. mar blue. We attempt to use the PU-DMPA for fabricating tissue engineering scaffolds. As electrospinningWe attempt to hasuse manythe PU-DMPA advantages for overfabricatin conventionalg tissue engineering methods in this scaffolds. regard As (e.g., elec- fibers trospinningwith the has capability many advantages of contact guidance), over conventi effortsonal were methods made in to this fabricate regard electrospun (e.g., fibers PU- withDMPA the capability fibrous membranes. of contact guidance), Despite the efforts relatively were low made molecular to fabricate weight electrospun of the PU-DMPA, PU- FigureDMPAbead 6. fibrous Growth free and membranes. of dropHUASMCs free Despite electrospun cultured the alone relatively fibers in tissue were low culture obtained molecular plates, when withweight the ofPU-BDO, concentration the PU-DMPA, PU- of the DMPA,beadPU-DMPA free and and gelatin-modified solutiondrop free was electrospun PU-DMPA, increased fibers torespectively 15% were (w/v, ). obtainedin The terms presence of when the percentage of the carboxyl concentration of groupsreduced in ofAla- thethe PU- marPU-DMPA DMPAblue. wassolution demonstrated was increased by immobilization to 15% (w/v). ofThe gelatin presence on the of as-spuncarboxyl PU-DMPA groups in fibrousthe PU-DMPAmembrane was using demonstrated carbodiimide by immobilization chemistry. The gelatinof gelatin treatment on the significantlyas-spun PU-DMPA decreased fi- the porosityWe attempt of as-spun to use the membranes PU-DMPA from for 78fabricatin± 3% tog 67tissue± 7%. engineering Figure7 shows scaffolds. SEM As images elec- of trospinningthe electrospun has many PU-DMPA advantages fibrous over membrane conventional and methods the gelatin-modified in this regard counterparts. (e.g., fibers The withdiameter the capability of gelatin-modified of contact guidance), fibers (451 efforts± 144 were nm) made was significantlyto fabricate electrospun greater than PU- that of DMPAas-spun fibrous fibers membranes. (380 ± 97 Despite nm) (p < the 0.05). relatively low molecular weight of the PU-DMPA, bead free and drop free electrospun fibers were obtained when the concentration of the PU-DMPA solution was increased to 15% (w/v). The presence of carboxyl groups in the PU-DMPA was demonstrated by immobilization of gelatin on the as-spun PU-DMPA fi-

Polymers 2021, 13, x FOR PEER REVIEW 14 of 20

brous membrane using carbodiimide chemistry. The gelatin treatment significantly decreased the porosity of as-spun membranes from 78 ± 3% to 67 ± 7%. Figure 7 shows SEM images of the electrospun PU-DMPA fibrous membrane and the gelatin-modified coun- Polymers 2021, 13, 1527 terparts. The diameter of gelatin-modified fibers (451 ± 144 nm) was significantly greater13 of 18 than that of as-spun fibers (380 ± 97 nm) (p < 0.05).

FigureFigure 7. 7. SEMSEM images images and and diameter diameter distribution distributionss of the of theas-spun as-spun PU-DMPA PU-DMPA fibers fibers (a–c) ( aand–c) andthe the gelatin-modifiedgelatin-modified PU-DMAP PU-DMAP fibers fibers (d (–df–);f );(b, (eb), eare) are at higher at higher magnifications. magnifications. The Thescale scale bar at bar the at the bottombottom left left of of SEM SEM images images is is 10 10 µm.µm.

FigureFigure 8 shows the FT-IR spectra of the gelatin-modifiedgelatin-modified PU-DMPA cast film,film, the gelatin-modifiedgelatin-modified electrospun electrospun PU-DMPA PU-DMPA membrane, membrane, and and a a negati negativeve control. control. The The negative negative controlcontrol was was prepared prepared by by simply simply placing placing a nona a nonactivatedctivated PU-DMPA PU-DMPA cast cast film film in gelatin in gelatin so- lutionsolution followed followed by bywashing washing with with deionized deionized water. water. The The characteristic characteristic peak peak of of the the gelatin gelatin −1 amideamide I Iat at 1645 1645 cm cm−1 andand the theH-bonded H-bonded O-H O-H stretching stretching broad broad peak peakextending extending from 3680 from −1 −1 3680−1 cm to 3150−1 cm indicate the successful immobilization of gelatin on the gelatin- Polymers 2021, 13, x FOR PEER REVIEWcm to 3150 cm indicate the successful immobilization of gelatin on the gelatin-modified15 of 20 specimens.modified specimens. There was Thereno difference was no differencebetween the between spectra the of spectra the negative of the negativecontrol and control the originaland the PU-DMPA original PU-DMPA cast film. castPonceau film. S Ponceau staining Salso staining confirmed also confirmedthe presence the of presence gelatin on of gelatin on the gelatin-modified specimens. the gelatin-modified specimens.

FigureFigure 8. 8.Left: Left schematic: schematic of of the the gelatin gelatin immobilization immobilization on the PU-DMPAPU-DMPA electrospunelectrospun fibers. fibers.Right Right:: ATR-FTIR ATR-FTIR spectra spectra of of the thePU-DMPA PU-DMPA solid solid film film (a ),(a the), the gelatin-modified gelatin-modified PU-DMPA PU-DMPA solid solid film film (b ),(b and), and the the gelatin-modified gelatin-modified PU-DMPA PU-DMPA electrospun electro- spunfibrous fibrous mesh mesh (c). The(c). The inserts inserts on the on rightthe right illustrate illustrate their their corresponding corresponding Ponceau Ponceau S staining. S staining.

ForFor more more functionalization functionalization routes ofof thethePU-DMPA, PU-DMPA, we we endowed endowed the the PU-DMPA PU-DMPA with withamino amino groups groups using using carbodiimide carbodiimide chemistry. chemistry. The aminoThe amino functionalization functionalization was performedwas performed on the PU-DMPA film surface or in a homogeneous solution of the PU-DMPA in THF. Figure 9 shows the FT-IR spectra of the PU-DMPA-BDA, the PU-DMPA-PLL, the PU-DMPA-BDA-HA, and the PU-DMPA-PLL-HA. The spectra of the PU-DMPA-BDA and the PU-DMPA-PLL showed the successful conjugation of amino groups either on the PU-DMPA film surface or in the PU-DMPA backbone. The presence of amino groups on the specimen surface was further confirmed by Ponceau S staining (data not shown). The spectra of the PU-DMPA-BDA-HA and the PU-DMPA-PLL-HA displayed a characteristic peak at 1645 cm−1, which is due to C=O stretching of an amide, and a broad peak centered about 3351 cm−1, which is attributed to profound H-bonded O-H stretching in HA, both indicating the successful grafting of HA. Upon the immobilization of HA, the contact angles of the PU-DMPA-BDA-HA (33.3 ± 4.3°) and the PU-DMPA-PLL-HA (28.6 ± 3.7°) cast films decreased significantly compared to that of the PU-DMPA-BDA (54.9 ± 4.0°) and PU- DMPA-PLL (54.0 ± 2.2°) films, respectively.

Polymers 2021, 13, 1527 14 of 18

on the PU-DMPA film surface or in a homogeneous solution of the PU-DMPA in THF. Figure9 shows the FT-IR spectra of the PU-DMPA-BDA, the PU-DMPA-PLL, the PU- DMPA-BDA-HA, and the PU-DMPA-PLL-HA. The spectra of the PU-DMPA-BDA and the PU-DMPA-PLL showed the successful conjugation of amino groups either on the PU-DMPA film surface or in the PU-DMPA backbone. The presence of amino groups on the specimen surface was further confirmed by Ponceau S staining (data not shown). The spectra of the PU-DMPA-BDA-HA and the PU-DMPA-PLL-HA displayed a characteristic peak at 1645 cm−1, which is due to C=O stretching of an amide, and a broad peak centered about 3351 cm−1, which is attributed to profound H-bonded O-H stretching in HA, both indicating the successful grafting of HA. Upon the immobilization of HA, the contact angles of the PU-DMPA-BDA-HA (33.3 ± 4.3◦) and the PU-DMPA-PLL-HA (28.6 ± 3.7◦) Polymers 2021, 13, x FOR PEER REVIEWcast films decreased significantly compared to that of the PU-DMPA-BDA (54.9 ± 4.0◦)16 and of 20

PU-DMPA-PLL (54.0 ± 2.2◦) ﬁlms, respectively.

FigureFigure 9. 9.Top: Top: schematic schematic of of the the amino amino functionalization functionalization of of the the PU-DMPA PU-DMPA cast cast film film and and bulk bulk polymer polymer solution by 1,4-BDA and PLL, respectively, followed by the incorporation of HA. Bottom: solution by 1,4-BDA and PLL, respectively, followed by the incorporation of HA. Bottom: ATR-FTIR ATR-FTIR spectra of the BDA-modified PU-DMPA (a), the PLL-modified PU-DMPA (b), the HA- spectra of the BDA-modified PU-DMPA (a), the PLL-modified PU-DMPA (b), the HA-modified modified PU-DMPA-BDA (c), and the HA-modified PU-DMPA-PLL (d). PU-DMPA-BDA (c), and the HA-modified PU-DMPA-PLL (d). The HA-modified surface of the PU-DMPA-BDA and the PU-DMPA-PLL films were The HA-modified surface of the PU-DMPA-BDA and the PU-DMPA-PLL films were subjectedsubjected to to platelet platelet adhesion adhesion tests tests withwith thethe PU-DMPA,PU-DMPA,PU-DMPA-BDA, PU-DMPA-BDA, and and PU-DMPA- PU-DMPA- PLLPLL films films serving serving as as controls. controls. A A material material that that has has antithrombotic antithrombotic ability ability might might be be applied applied inin the the developmentdevelopment of blood blood contact contact devices. devices. Figure Figure 10 10 shows shows the the SEM SEM images images of the of mor- the phology of platelets adhered on the five surfaces. SEM showed less platelet adhesion on the surface of the PU-DMPA-BDA-HA and the PU-DMPA-PLL-HA films. The attached platelets, if any, had round shapes. The PU-DMPA-PLL-HA membrane even had less platelet adhesion than the PU-DMPA-BDA-HA. The results may be attributed to more amino groups in PLL and hence more HA conjugation on the PU-DMPA-PLL. Despite the limited number of carboxyl groups in the PU-DMPA backbone, the number of available amino groups was multiplied with the use of PLL as a linker. Obvious platelet adhesion on the PU-DMPA-BDA and the PU-DMPA-PLL was noted possibly due to the positive charge of amino groups. Interestingly, the PU-DMPA surface also displayed only few adhered platelets, perhaps due to the negative charged of the carboxyl groups. Hong et al. also found reduced platelet adhesion on the surface of their poly(ester urethane) bearing carboxyl groups [18]. This contradicts the finding in an earlier study; however, note that hydroxytelechelic polybutadiene was used for soft segments in said study [38].

Polymers 2021, 13, 1527 15 of 18

morphology of platelets adhered on the five surfaces. SEM showed less platelet adhesion on the surface of the PU-DMPA-BDA-HA and the PU-DMPA-PLL-HA films. The attached platelets, if any, had round shapes. The PU-DMPA-PLL-HA membrane even had less platelet adhesion than the PU-DMPA-BDA-HA. The results may be attributed to more amino groups in PLL and hence more HA conjugation on the PU-DMPA-PLL. Despite the limited number of carboxyl groups in the PU-DMPA backbone, the number of available amino groups was multiplied with the use of PLL as a linker. Obvious platelet adhesion on the PU-DMPA-BDA and the PU-DMPA-PLL was noted possibly due to the positive charge of amino groups. Interestingly, the PU-DMPA surface also displayed only few adhered platelets, perhaps due to the negative charged of the carboxyl groups. Hong et al. also found reduced platelet adhesion on the surface of their poly(ester urethane) bearing Polymers 2021, 13, x FOR PEER REVIEW 17 of 20 carboxyl groups [18]. This contradicts the finding in an earlier study; however, note that hydroxytelechelic polybutadiene was used for soft segments in said study [38].

Figure 10. SEM images of platelets adhered onto the surface of the PU-DMPA (a), the BDA-modified Figure 10. SEM images of platelets adhered onto the surface of the PU-DMPA (a), the BDA-modi- fiedPU-DMPA PU-DMPA ( b(b),), the the PLL-modifiedPLL-modified PU-DMPAPU-DMPA (c), the the HA-modified HA-modified PU-DMPA-BDA PU-DMPA-BDA (d (d),), and and the theHA-modified HA-modified PU-DMPA-PLLPU-DMPA-PLL ( e(e).).

CautionCaution must must be betaken taken as as surface surface modification modification allows allows imparting imparting materials withwith specificspe- cificfunctions, functions, but but the the effect effect fadesfades outout as th thee material material surface surface degrades. degrades. Incorporation Incorporation of of pendantpendant reactive reactive groups groups by bybulk bulk modification modification for for subsequent subsequent functionalization functionalization would would be be more endurable. In this study, both approaches were performed to amino-functionalize the more endurable. In this study, both approaches were performed to amino-functionalize PU-DMPA. The immobilization of HA on the amino-functionalized PU-DMPA film was the PU-DMPA. The immobilization of HA on the amino-functionalized PU-DMPA film performed by surface modification, however. was performed by surface modification, however. There are a few limitations in this study. The number of carboxyl groups in the PU- There are a few limitations in this study. The number of carboxyl groups in the PU- DMPA was determined by the DMPA content. In this study, isocyanate-terminated PCL DMPA was determined by the DMPA content. In this study, isocyanate-terminated PCL prepolymer was formed prior to the reaction with equimolar amount of DMPA, which prepolymer was formed prior to the reaction with equimolar amount of DMPA, which resulted in an alternating copolymer—that is, the carboxyl groups were evenly distributed resulted in an alternating copolymer—that is, the carboxyl groups were evenly distributed in the PU-DMPA backbone. We did not attempt to manipulate the DMPA content by mixing in the PU-DMPA backbone. We did not attempt to manipulate the DMPA content by mix- PCL diol with DMPA when forming the prepolymer nor by changing the ratio of PCL diol ing PCL diol with DMPA when forming the prepolymer nor by changing the ratio of PCL and DMPA chain extender, which could result in block copolymers with unpredictable diol and DMPA chain extender, which could result in block copolymers with unpredicta- structures. The changes in the DMPA content might increase the functionalizability of the ble structures. The changes in the DMPA content might increase the functionalizability of the polyurethane but could alter the mechanical properties of the polyurethane, which deserves a systematic investigation. It was suggested an increase in the DMPA content could reduce the molecular weight of the polymer [18], however. Note that polymer chain entanglement in solutions, which is closely associated with the molecular weight of a polymer, is an important prerequisite for electrospinning of the polymer. The carboxyl group in DMPA may react with isocyanates, although the steric hindrance that is due to the methyl group in DMPA could impede this reaction [39]. Using chain extender with a protecting group may reduce the consumption of carboxyl groups during synthesis [40]. The toxicity of tin-based catalysts remains a concern despite no obvious cytotoxicity being observed. Sn(Oct)2 may be replaced by amine catalysts such as triethylenediamine, which can be removed by water. Finally, due to the improved hydrophilicity of the PU-DMPA compared with the PU-BDO, the degradation rate of the PU-DMPA might be increased.

Polymers 2021, 13, 1527 16 of 18

polyurethane but could alter the mechanical properties of the polyurethane, which deserves a systematic investigation. It was suggested an increase in the DMPA content could reduce the molecular weight of the polymer [18], however. Note that polymer chain entanglement in solutions, which is closely associated with the molecular weight of a polymer, is an important prerequisite for electrospinning of the polymer. The carboxyl group in DMPA may react with isocyanates, although the steric hindrance that is due to the methyl group in DMPA could impede this reaction [39]. Using chain extender with a protecting group may reduce the consumption of carboxyl groups during synthesis [40]. The toxicity of tin-based catalysts remains a concern despite no obvious cytotoxicity being observed. Sn(Oct)2 may be replaced by amine catalysts such as triethylenediamine, which can be removed by water. Finally, due to the improved hydrophilicity of the PU-DMPA compared with the PU-BDO, the degradation rate of the PU-DMPA might be increased. The biodegradability of the PU-DMPA compared to the PU-BDO may be worth a thorough investigation.

4. Conclusions The PU-DMPA, based on a PCL soft segment, a HDI hard segment and a DMPA chain extender, was synthesized successfully as designed. The time required to complete the chain extension of the PU-DMPA was tremendously longer than that of the control PU-BDO (4 days vs. 24 h) and the PU-DMPA had a much lower molecular weight than the PU-BDO. The melting enthalpy of the PU-DMPA was greater than that of the PU-BDO. The PU-DMPA had a higher initial modulus and a lower linear modulus and ultimate strength than the PU-BDO, whereas the elongation at break of both the polyurethanes were comparable. The PU-DMPA was more hydrophilic than the PU-BDO. The different properties and behaviors might be attributed to the carboxyl groups in the PU-DMPA. Both the polyurethanes exhibited no cytotoxicity. The improved hydrophilicity of the PU-DMPA manifested better cell adhesion. Bead free and drop free electrospun PU-DMPA fibrous membranes with a narrow fiber diameter distribution were successfully fabricated using a relatively concentrated PU-DMPA solution. Because of the presence of carboxyl groups in the PU-DMPA backbone, compounds containing amino groups can be conjugated to the PU-DMPA using carbodiimide chemistry. Compounds containing carboxyl groups, on the other hand, can be immobilized to the amino-functionalized PU-DMPA. Gelatin was successfully conjugated to the electrospun PU-DMPA membrane using carbodiimide chemistry. HA was immobilized on the amino- functionalized PU-DMPA cast film. Reduced platelet adhesion of the HA-modified PU- DMPA was noted. The PU-DMPA has the potential to be used as a scaffold material for soft tissue engineering.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/polym13091527/s1, Figure S1: GPC chromatograms of the PU-DMPA and the PU-BDO. Author Contributions: Conceptualization, J.-J.H. and C.-C.L.; methodology, J.-J.H. and C.-C.L.; investigation, C.-C.L.; formal analysis, C.-C.L., C.-H.L. and H.-Y.T.-M.; validation, J.-J.H.; visualization, J.-J.H. and C.-C.L.; resources, J.-J.H.; writing—original draft preparation, J.-J.H. and C.-H.L.; writing— review and editing, J.-J.H.; supervision, J.-J.H.; funding acquisition, J.-J.H. All authors have read and agreed to the published version of the manuscript. Funding: Financial supports from Taipei Veterans General Hospital (V110B-011), the Ministry of Science and Technology (MOST-109-2221-E-009-024) and the National Health Research Institutes (NHRI-EX109-10827EI) in Taiwan are gratefully acknowledged. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available on request from the corresponding author. Acknowledgments: We are grateful to Kuo-Huang Hsieh and Shenghong Dai for advice on the synthesis of polyurethanes. Polymers 2021, 13, 1527 17 of 18

Conﬂicts of Interest: The authors declare no conﬂict of interest.

References 1. Choi, J.H.; Gimble, J.M.; Lee, K.; Marra, K.G.; Rubin, J.P.; Yoo, J.J.; Vunjak-Novakovic, G.; Kaplan, D.L. Adipose Tissue Engineering for Soft Tissue Regeneration. Tissue Eng. Part B Rev. 2010, 16, 413–426. [CrossRef] 2. Colen, D.L.; Carney, M.J.; Shubinets, V.; Lanni, M.A.; Liu, T.; Levin, L.S.; Lee, G.C.; Kovach, S.J. Soft-Tissue Reconstruction of the Complicated Knee Arthroplasty: Principles and Predictors of Salvage. Plast. Reconstr. Surg. 2018, 141, 1040–1048. [CrossRef] 3. Marler, J.J.; Upton, J.; Langer, R.; Vacanti, J.P. Transplantation of cells in matrices for tissue regeneration. Adv. Drug Deliv. Rev. 1998, 33, 165–182. [CrossRef] 4. Jagur-Grodzinski, J. Biomedical application of functional polymers. React. Funct. Polym. 1999, 39, 99–138. [CrossRef] 5. Nair, L.S.; Laurencin, C.T. Biodegradable polymers as biomaterials. Prog. Polym. Sci. 2007, 32, 762–798. [CrossRef] 6. Gunatillake, P.; Mayadunne, R.; Adhikari, R. Recent developments in biodegradable synthetic polymers. Biotechnol. Annu. Rev. 2006, 12, 301–347. [CrossRef] 7. Wang, Y.D.; Ameer, G.A.; Sheppard, B.J.; Langer, R. A tough biodegradable elastomer. Nat. Biotechnol. 2002, 20, 602–606. [CrossRef] 8. Yang, J.; Webb, A.R.; Ameer, G.A. Novel citric acid-based biodegradable elastomers for tissue engineering. Adv. Mater. 2004, 16, 511–516. [CrossRef] 9. You, Z.R.; Hu, M.H.; Tuan-Mu, H.Y.; Hu, J.J. Fabrication of poly(glycerol sebacate) fibrous membranes by coaxial electrospinning: Influence of shell and core solutions. J. Mech. Behav. Biomed. 2016, 63, 220–231. [CrossRef] 10. Jeffries, E.M.; Allen, R.A.; Gao, J.; Pesce, M.; Wang, Y.D. Highly elastic and suturable electrospun poly(glycerol sebacate) fibrous scaffolds. Acta Biomater. 2015, 18, 30–39. [CrossRef] 11. Wu, H.J.; Hu, M.H.; Tuan-Mu, H.Y.; Hu, J.J. Preparation of aligned poly(glycerol sebacate) fibrous membranes for anisotropic tissue engineering. Mater. Sci. Eng. C 2019, 100, 30–37. [CrossRef] 12. Place, E.S.; George, J.H.; Williams, C.K.; Stevens, M.M. Synthetic polymer scaffolds for tissue engineering. Chem. Soc. Rev. 2009, 38, 1139–1151. [CrossRef][PubMed] 13. Liu, Q.Y.; Jiang, L.; Shi, R.; Zhang, L.Q. Synthesis, preparation, in vitro degradation, and application of novel degradable bioelastomers-A review. Prog. Polym. Sci. 2012, 37, 715–765. [CrossRef] 14. Davis, F.J.; Mitchell, G.R. Polyurethane based materials with applications in medical devices. In Bio-Materials and Prototyping Applications in Medicine; Springer: Berlin/Heidelberg, Germany, 2008; pp. 27–48. 15. Gong, F.R.; Lu, Y.; Guo, H.; Cheng, S.J.; Gao, Y. Hyaluronan Immobilized Polyurethane as a Blood Contacting Material. Int. J. Polym. Sci. 2010, 2010, 807935. [CrossRef] 16. Chen, J.; Dong, R.N.; Ge, J.; Guo, B.L.; Ma, P.X. Biocompatible, Biodegradable, and Electroactive Polyurethane-Urea Elastomers with Tunable Hydrophilicity for Skeletal Muscle Tissue Engineering. ACS Appl. Mater. Interfaces 2015, 7, 28273–28285. [CrossRef] 17. Mondal, S.; Martin, D. Hydrolytic degradation of segmented polyurethane copolymers for biomedical applications. Polym. Degrad. Stabil. 2012, 97, 1553–1561. [CrossRef] 18. Hong, Y.; Ye, S.H.; Pelinescu, A.L.; Wagner, W.R. Synthesis, Characterization, and Paclitaxel Release from a Biodegradable, Elastomeric, Poly(ester urethane)urea Bearing Phosphorylcholine Groups for Reduced Thrombogenicity. Biomacromolecules 2012, 13, 3686–3694. [CrossRef][PubMed] 19. Song, Q.J.; Chen, H.M.; Zhou, S.B.; Zhao, K.Q.; Wang, B.Q.; Hu, P. Thermo- and pH-sensitive shape memory polyurethane containing carboxyl groups. Polym. Chem. 2016, 7, 1739–1746. [CrossRef] 20. Griffin, M.; Castro, N.; Bas, O.; Saifzadeh, S.; Butler, P.; Hutmacher, D.W. The Current Versatility of Polyurethane Three- Dimensional Printing for Biomedical Applications. Tissue Eng. Part B Rev. 2020, 26, 272–283. [CrossRef][PubMed] 21. Guelcher, S.A. Biodegradable polyurethanes: Synthesis and applications in regenerative medicine. Tissue Eng. Part B Rev. 2008, 14, 3–17. [CrossRef] 22. Pedicini, A.; Farris, R.J. Mechanical behavior of electrospun polyurethane. Polymer 2003, 44, 6857–6862. [CrossRef] 23. Bhardwaj, N.; Kundu, S.C. Electrospinning: A fascinating fiber fabrication technique. Biotechnol. Adv. 2010, 28, 325–347. [CrossRef] 24. Hu, J.J.; Chao, W.C.; Lee, P.Y.; Huang, C.H. Construction and characterization of an electrospun tubular scaffold for small-diameter tissue-engineered vascular grafts: A scaffold membrane approach. J. Mech. Behav. Biomed. 2012, 13, 140–155. [CrossRef][PubMed] 25. Reneker, D.H.; Yarin, A.L.; Fong, H.; Koombhongse, S. Bending instability of electrically charged liquid jets of polymer solutions in electrospinning. J. Appl. Phys. 2000, 87, 4531–4547. [CrossRef] 26. Pawlowska, S.; Rinoldi, C.; Nakielski, P.; Ziai, Y.; Urbanek, O.; Li, X.R.; Kowalewski, T.A.; Ding, B.; Pierini, F. Ultraviolet Light-Assisted Electrospinning of Core-Shell Fully Cross-Linked P(NIPAAm-co-NIPMAAm) Hydrogel-Based Nanofibers for Thermally Induced Drug Delivery Self-Regulation. Adv. Mater. Interfaces 2020, 7, 2000247. [CrossRef] 27. Nakielski, P.; Pawlowska, S.; Rinoldi, C.; Ziai, Y.; De Sio, L.; Urbanek, O.; Zembrzycki, K.; Pruchniewski, M.; Lanzi, M.; Salatelli, E.; et al. Multifunctional Platform Based on Electrospun Nanofibers and Plasmonic Hydrogel: A Smart Nanostructured Pillow for Near-Infrared Light-Driven Biomedical Applications. ACS Appl. Mater. Interfaces 2020, 12, 54328–54342. [CrossRef][PubMed] Polymers 2021, 13, 1527 18 of 18

28. De Sio, L.; Ding, B.; Focsan, M.; Kogermann, K.; Pascoal-Faria, P.; Petronela, F.; Mitchell, G.; Zussman, E.; Pierini, F. Personalized Reusable Face Masks with Smart Nano-Assisted Destruction of Pathogens for COVID-19: A Visionary Road. Chem. Eur. J. 2021, 27, 6112–6130. [CrossRef][PubMed] 29. Li, C.Y.; Hu, M.H.; Hu, J.J. Use of Aligned Microscale Sacrificial Fibers in Creating Biomimetic, Anisotropic Poly(glycerol sebacate) Scaffolds. Polymers 2019, 11, 1492. [CrossRef][PubMed] 30. Coleman, M.M.; Lee, K.H.; Skrovanek, D.J.; Painter, P.C. Hydrogen-Bonding in Polymers.4. Infrared Temperature Studies of a Simple Polyurethane. Macromolecules 1986, 19, 2149–2157. [CrossRef] 31. Jiang, L.; Ren, Z.Y.; Zhao, W.; Liu, W.T.; Liu, H.; Zhu, C.S. Synthesis and structure/properties characterizations of fou polyurethane model hard segments. R. Soc. Open Sci. 2018, 5, 180536. [CrossRef] 32. Wang, C.B.; Cooper, S.L. Morphology and Properties of Segmented Polyether Polyurethaneureas. Macromolecules 1983, 16, 775–786. [CrossRef] 33. Guan, J.J.; Sacks, M.S.; Beckman, E.J.; Wagner, W.R. Synthesis, characterization, and cytocompatibility of efastomeric, biodegradable poly(ester-urethane)ureas based on poly(caprolactone) and putrescine. J. Biomed. Mater. Res. 2002, 61, 493–503. [CrossRef] [PubMed] 34. Mamidi, N.; Zuniga, A.E.; Villela-Castrejon, J. Engineering and evaluation of forcespun functionalized carbon nano-onions reinforced poly (epsilon-caprolactone) composite nanofibers for pH-responsive drug release. Mater. Sci. Eng. C 2020, 112, 110928. [CrossRef][PubMed] 35. Griffin, M.; Premakumar, Y.; Seifalian, A.; Butler, P.E.; Szarko, M. Biomechanical Characterization of Human Soft Tissues Using Indentation and Tensile Testing. JoVE J. Vis. Exp. 2016, e54872. [CrossRef][PubMed] 36. Ebrahimi, A.P. Mechanical properties of normal and diseased cerebrovascular system. J. Vasc. Interv. Neurol. 2009, 2, 155–162. 37. van Wachem, P.B.; Beugeling, T.; Feijen, J.; Bantjes, A.; Detmers, J.P.; van Aken, W.G. Interaction of cultured human endothelial cells with polymeric surfaces of different wettabilities. Biomaterials 1985, 6, 403–408. [CrossRef] 38. Poussard, L.; Burel, F.; Couvercelle, J.P.; Merhi, Y.; Tabrizian, M.; Bunel, C. Hemocompatibilty of new ionic polyurethanes: Influence of carboxylic group insertion modes. Biomaterials 2004, 25, 3473–3483. [CrossRef] 39. Zhang, S.B.; Miao, W.; Zhou, Y. Reaction study of water-borne polyurethanes based on isophorone diisocyanate, dimethylol propionic acid, and poly(hexane neopentyl adipate glycol). J. Appl. Polym. Sci. 2004, 92, 161–164. [CrossRef] 40. Fang, J.; Zhang, J.L.; Du, J.; Pan, Y.J.; Shi, J.; Peng, Y.X.; Chen, W.M.; Yuan, L.; Ye, S.H.; Wagner, W.R.; et al. Orthogonally Functionalizable Polyurethane with Subsequent Modification with Heparin and Endothelium-Inducing Peptide Aiming for Vascular Reconstruction. ACS Appl. Mater. Interfaces 2016, 8, 14442–14452. [CrossRef][PubMed]