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Thermal annealing to modulate the shape memory behavior of a biobased and biocompatible triblock copolymer scaffold in the human body temperature range Andrea Merlettini, Matteo Gigli, Martina Ramella, Chiara Gualandi, Michelina Soccio, Francesca Boccafoschi, Andrea Munari, Nadia Lotti, and Maria Letizia Focarete Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.7b00644 • Publication Date (Web): 21 Jun 2017 Downloaded from http://pubs.acs.org on June 25, 2017

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Thermal annealing to modulate the shape memory behavior of a biobased and biocompatible triblock copolymer scaffold in the human body temperature range

Andrea Merlettini,a,‡ Matteo Gigli,b,‡,† Martina Ramella,c Chiara Gualandi,a,d Michelina Soccio,b Francesca Boccafoschi,c Andrea Munari,b Nadia Lotti,b Maria Letizia Focaretea,d* a

Department of Chemistry “G. Ciamician” and INSTM UdR of Bologna, University of Bologna, via

Selmi 2, 40126 Bologna, Italy b

Department of Civil, Chemical, Environmental and Materials Engineering, University of Bologna,

via Terracini 28, 40131 Bologna, Italy c

Department of Health Sciences, University of Piemonte Orientale, via Solaroli 17, 28100 Novara,

Italy d

Health Sciences and Technologies and Interdepartmental Center for Industrial Research (HST-

ICIR), University of Bologna, via Tolara di Sopra 41/E 40064, Ozzano dell'Emilia, Bologna, Italy

KEYWORDS: Shape Memory Polymers; Electrospinning; Block copolymers; Polylactic acid; Thermal annealing; Physiological temperature

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ABSTRACT

A biodegradable and biocompatible electrospun scaffold with shape memory behavior in the physiological temperature range is here presented. It was obtained starting from a specifically designed, biobased PLLA-based triblock copolymer, where the central block is poly(propylene azelate-co-propylene sebacate), (P(PAz60PSeb40)) random copolymer. Shape memory properties are determined by the contemporary presence of the low melting crystals of the P(PAz60PSeb40) block, acting as switching segment, and of the high melting crystal phase of PLLA blocks, acting as physical network. It is demonstrated that a straightforward annealing process applied to the crystal phase of the switching element gives the possibility to tune the shape recovery temperature from about 25°C to 50°C, without the need of varying the copolymer’s chemical structure. The thermal annealing approach here presented can be thus considered a powerful strategy for ‘ad hoc’ programming the same material for applications requiring different recovery temperatures. Fibroblast culture experiments demonstrated scaffold biocompatibility.

INTRODUCTION Shape Memory Polymers (SMPs) are smart, stimuli-responsive materials that have received increasing attention in the last years for different applications where their unique properties are profitably exploited:1-12 they can be deformed from a permanent to a temporary shape and are capable to recover the original, permanent shape through an external stimulus, such as temperature,13 light,14 or electrical and magnetic fields.15 In order to exhibit shape memory (SM) behavior, the polymer microstructure must possess: (i) switching domains capable to respond to the external triggering stimulus, that enable to fix the temporary shape, and (ii) a stable network of permanent domains formed by chemically or physically crosslinked structures, known as net-points, that is responsible of the recovery of the permanent shape.


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The most common SMPs are activated by thermal stimuli7,13 and make use of polymer thermal transitions. Depending on the solid-state characteristic of the switching phase (either amorphous or semicrystalline), the glass transition (Tg) or the melting (Tm) temperatures can be chosen as the shape transition temperature (Ttrans). It is pointed out that Tm is advantageously used as shape transition temperature since it is a 1st order transition, thus not affected by kinetics effects, it generally occurs over a smaller temperature range than the glass transition and its width can be regulated by annealing treatments. In a context where the thermal transitions are the key elements of SM materials, among the different macromolecular structures with SM properties that have been explored,2 copolymer systems are of great interest given the possibility to tune their properties by playing on the copolymer composition and microstructure. In particular, in block copolymers, differently from random copolymers, the critical length of the individual blocks to develop a crystalline phase is generally reached and phase segregation between hard segments (related to the stable network) and soft segments (associated with the switching phase) occurs. Such copolymer systems have been thoroughly investigated in the literature, among which: (i) polyurethane (PU)-based SM copolymers, whose properties can be tailored by reacting a wide range of diamine or dihydroxyterminated oligomers with diisocyanates;3,4,16,17 (ii) segmented (multi)block copolymers with poly(ethylene oxide) (PEO) as soft segment in a block copolymer with poly(ethylene terephthalate) (PET),18 as soft block in PU,19 in crosslinked polymethacrylate networks,20 as well as in poly(pdioxanone)-poly(ethylene glycol) systems;21 (iii) thermoplastic elastomer block copolymers of polylactones,17,22 and in particular of low melting aliphatic polyesters such as poly(ε-caprolactone) (PCL), used as soft blocks in PU systems,3,4 with the possibility to achieve low switching temperatures (closed to the human body temperature) by controlling the molecular weight of the polyester soft block.23


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Among thermoplastic block copolymers, lactide-based SMPs are attracting increasing attention, given their biocompatibility, biodegradability, and biobased character. A wide variety of SM lactide-based copolymers have been synthesized so far.24 However, owing to the low activity of the lactic acid monomer, the direct condensation of the acid usually results in low-molecular-weight copolymers. Therefore, high molecular weight polymers are often achieved through chain-extension reactions, obtained either from direct polycondensation or ring-opening polymerization of lactide, with hexamethylene diisocyanate (HDI) as the chain extender.25-27 Biocompatible SM polymeric materials gathered increasing interest in the medical field as biomedical devices28,29 for applications in minimally invasive surgery procedures, facilitating the manipulation of surgical aids, such as intelligent sutures, or the insertion of implants such as stents and catheters.29 Moreover, SMPs have also gained interest in the field of tissue engineering as a promising approach for creating smart tissue engineered scaffolds to promote regeneration of tissues and organs. A number of research papers described the use of SM polymeric scaffolds as substrates for cell growth.30-34 In this framework, besides allowing minimally invasive surgical implantation, the SMP scaffold can possess multiple functionalities such as the ability to regulate cell behavior by exerting appropriate mechanical stimuli or by changing substrate topography.30 Most of the tissue engineered applications of SMPs make use of micro/nano fibrous scaffolds that can be successfully fabricated by means of the electrospinning technology,35-43 obtaining, in some cases, scaffolds that displayed an enhanced SM effect than the corresponding bulk film.37 Indeed, it is well known that micro/nano-structuring has a tremendous influence on materials properties and functionality, that are usually promoted and amplified. For these reasons, SM micro/nano-structured scaffolds obtained by electrospinning technology have received increasing attention in the last years. A recent example regards the development of a multifunctional fibrous scaffold based on poly(D,L-lactide) and poly(trimethylene carbonate), which combines the capability to mimic the bone tissue architecture, showing good osteoblast proliferation, with SM effect demonstrated by 4 ACS Paragon Plus Environment

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excellent shape fixity and shape recovery rates.35 In another interesting study, a thermoresponsive electrospun scaffold was fabricated by electrospinning a SMP that was programmed to change macroscopic shape and fiber alignment during cell culture. The obtained scaffold was used to test the hypothesis that a shape memory-actuated modification of scaffold microscopic structure could control the behavior of adherent cells.42 We recently reported the two-way shape memory behavior of an electrospun PCL-based scaffold capable of reversibly changing its shape between two distinguished configurations on the application of on/off stimuli in the range 30-60°C.43 To the success of the biomedical device the SM transition should be as closest as possible to the physiological temperature. Several strategies have been endeavored to achieve control of the shapememory trigger temperature. Among these, the melting temperature of poly(ester urethane)s has been reduced by introducing low molecular weight crystallizable soft segments13 or, as mentioned above, by controlling the size of phase-segregated hard segment domains,44,45 or designing semicrystalline networks with architectures that hinder crystallization, thus depressing crystallization and melting transitions.46 This work aims at contributing to the field of SMPs by providing additional knowledge on both the fundamental correlation between microstructure and shape memory properties and on the practical application of the designed material. In this research, a new linear triblock copolymer was specifically designed and synthesized to gain a biodegradable and biocompatible polymeric material showing a thermally induced shape-memory effect in the physiological temperature range. This effect was achieved thanks to the concomitant presence of a low melting crystal phase acting as switching segment, with the capability to promptly fix the temporary shape, and of a high melting crystal phase acting as physical network. An additional interesting feature is the strong biobased character of the polymer, synthesized starting from lactic acid, 1,3-propanediol, azelaic acid and sebacic acid. The copolymer was used to fabricate an electrospun shape memory scaffold, activated in the range of human body T, intended for biomedical applications, whose biocompatibility was 5 ACS Paragon Plus Environment

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evaluated by fibroblast cell culture. Moreover, in this work we show for the first time that a thermal annealing step, applied to the thermo-mechanical cycle after the fixing step, gives the possibility to control the shape-memory trigger temperature.

EXPERIMENTAL SECTION Materials. Sebacic acid and azelaic acid (TCI Chemicals), 1,3-propanediol, titanium tetrabutoxide (Ti(OBu)4), Tin(II)-2-ethylhexanoate (Sn(Oct)2) and hexamethylene diisocyanate (HDI) (Sigma-Aldrich) were reagent grade products. L-Lactide(99%) was kindly provided by Corbion. All products were used as supplied, with the exception of Ti(OBu)4 and Sn(Oct)2 that were distilled before use. Methanol, chloroform, dichloromethane (DCM) and N,N-dimethylformamide (DMF) were purchased from Sigma-Aldrich and used without any further purification. Polymer synthesis. The synthesis of the high molecular weight triblock copolymer poly(L-lactic acid)- poly(propylene azelate-co-propylene sebacate)-poly(L-lactic acid) [PLLA- P(PAz60PSeb40)PLLA], herein defined HMW-ABA, involved three steps, as outlined in Scheme 1. Synthesis of the random hydroxyl-terminated prepolymer. Random hydroxyl-terminated poly(propylene azelate-co-propylene sebacate) (60/40), P(PAz60PSeb40), herein defined B block, was synthesized by reacting 1,3-propanediol with a mixture of the two dicarboxylic acids: 60 mol% azelaic and 40 mol% sebacic (Scheme 1, I step). To obtain an OH-terminated prepolymer, an excess (60%) of 1,3-propanediol with respect to the dicarboxylic acids was employed. The reaction was carried out in bulk according to the standard melt polycondensation procedure by employing Ti(OBu)4 as catalyst (about 150 ppm of Ti/g of polymer). The first part was run at 170°C under nitrogen flux until over 90% of the theoretical water distilled off (about 70 min). In the second part the temperature was increased to 220°C and the pressure was gradually reduced to 0.01 mbar to aid the removal of the excess of glycol. The reaction was stopped after additional 120 min. The 6 ACS Paragon Plus Environment

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obtained B block was purified by dissolution in chloroform and reprecipitation in methanol and was dried at room temperature (RT) under vacuum for at least 7 days prior to further use. Synthesis of the chain extended triblock copolymer (HMW-ABA). The PLLA-P(PAz60PSeb40)PLLA triblock copolymer (ABA) was synthesized by ring-opening polymerization (ROP) of Llactide, by using P(PAz60PSeb40) prepolymer as initiator (L-lactide: P(PAz60PSeb40) ratio equal to 4:6 by weight) and Sn(Oct)2 (100 ppm/g of polymer) as catalyst (Scheme 1, II step). The reaction was carried out in the melt at 170°C under nitrogen flux. To further increase the molecular weight of the triblock copolymer, HDI has been used as chain extender, thus leading to the formation of urethane bonds. An equimolar amount of HDI with respect to the OH-terminal groups of P(PAz60PSeb40), as measured by 1H-NMR, was added to the reaction mixture after about 3h (Scheme 1, III step). The temperature was set to 160°C and the reaction was allowed to continue for another 45 min. The chain extended ABA copolymer, hereafter referred to as HMW-ABA, was purified by dissolution in chloroform and reprecipitation in methanol and was dried at RT under vacuum for at least 7 days prior to further characterization.


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Scheme 1. Reaction scheme for the synthesis of the HMW-ABA copolymer.

Electrospun scaffolds preparation. The obtained HMW-ABA was dissolved at a concentration of 25% w/v in DCM:DMF = 65:35 (v/v) at RT. The solution was then transferred to a glass syringe and electrospun by means of a electrospinning apparatus (Spinbow srl, Italy), comprised of a highvoltage power supply, a syringe pump, a stainless steel blunt-ended needle (inner diameter 0.84 mm) connected with the power supply electrode (∆V = 20 kV), and a grounded aluminum drumtype collector (diameter 5 cm) rotating at 75 rpm. Polymer solution was dispensed with a flow rate of 1.2 mL/h through a PTFE tube to the needle, which was placed vertically on the collecting drum at a distance of 20 cm. All scaffolds were produced by electrospinning the polymeric solution for a period of 2 hours, to gain samples with thickness in the range 150-170 µm. The electrospun scaffolds were produced at RT and at relative humidity of 30%. 8 ACS Paragon Plus Environment

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Characterization techniques. Polymer structure and composition were determined by means of proton nuclear magnetic resonance (1H-NMR) spectroscopy at RT, employing a Varian Inova 400MHz instrument (Agilent Technologies). Deuterated chloroform was used as solvent (sample concentration of about 15 mg/mL). Molecular weights were evaluated by gel permeation chromatography (GPC) at 30 °C using a 1100 HPLC system (Agilent Technologies) equipped with PLgel 5-mm MiniMIX-C column (Agilent Technologies). A refractive index was employed as detector. Chloroform was used as eluent with a 0.3 mL/min flow and sample concentrations of about 2 mg/mL. A molecular weight calibration curve was obtained with polystyrene standards in the range 2000-100000 g/mol. The concentration of the carboxyl end-groups of the P(PAz60PSeb40) prepolymer was determined by titration. About 0.1 g of polymer was dissolved in chloroform at RT and the solution was titrated by using a standard NaOH in methanol and phenol red as indicator. Thermogravimetric analysis (TGA) was run under nitrogen atmosphere using a PerkinElmer TGA7 apparatus (gas flow: 10 mL/min) at 10°C/min, up to 800 °C. The temperatures relative to the onset of the degradation process (Tonset) and to the maximum weight loss rate (Tmax) were determined. Thermal transitions were measured by means of a differential scanning calorimeter (DSC Q100; TA Instruments, New Castle, Delaware, USA), equipped with a liquid nitrogen cooling system (LNCS) low temperature accessory. About 5 mg of sample were placed in aluminum pans and subjected to a heating scan at 20 °C/min from -100 °C to +200°C, quenched to 100 °C, and then heated up to 200 °C at 20°C/min, under helium atmosphere. Glass transition temperature (Tg) values were taken at half-height of the glass transition heat capacity steps while the cold crystallization temperature (Tcc) and the melting temperature (Tm) were taken at the peak maximum of crystallization exotherm and melting endotherm respectively. Samples annealed either at 15°C or 30°C in the Dynamic Mechanical Analyzer under a constrained strain (see below) were quickly transferred in the aluminum pans at 0°C and then analyzed by means of DSC from -100°C to 60°C at a heating rate of 3°C/min. DSC was also used in the temperature modulated mode (TMDSC): the sample was analyzed in the temperature range -80°C to +70°C at a heating rate of 3 9 ACS Paragon Plus Environment

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°C/min, whereas oscillation amplitude and oscillation period were 0.32 °C and 40 s, respectively. Wide angle X-ray diffraction measurements (WAXD) were carried out at RT with a PANalyticalX’Pert PRO diffractometer equipped with an XCelerator detector. A Cu anode was used as X-ray source (λ1 = 0.15406 nm, λ2 = 0.15443 nm). Fiber morphology was observed with a Philips 515 scanning electron microscope (SEM) at an accelerating voltage of 15 kV. Prior to SEM analysis, samples were sputter-coated with gold. The distribution of fiber diameters was determined through the measurement of about 300 fibers by means of an acquisition and image analysis software (EDAX Genesis) and the results were given as the average diameter ± standard deviation (SD). Shape memory properties characterization. The shape memory behavior of the electrospun scaffolds was studied using a DMA Q800 (TA Instruments, NewCastle, Delaware, USA) in tensile configuration. The specimens were cut as rectangular strips (overall length: 30 mm; width: 0.5 mm), heated at 50 °C and allowed to shrink until constant length was obtained. This treatment was necessary to completely melt the B block and to stabilize the dimension of the specimens, since electrospun samples shrunk when heated at 50°C (i.e. above the Tm of the B block), as a consequence of relaxation of the oriented chain segments that experienced high stretching during the electrospinning process.47,48 Shape memory behavior of the electrospun samples was assessed, soon after the shrinkage, through a three-step thermo-mechanical loading–unloading cycle: (i) cool the sample to 30°C and apply deformation (εappl) of about 150% under load control (loading rate of 0.5 N/min) to get the temporary shape (programming step); (ii) fix the temporary shape by cooling the sample to -60°C under fixed strain conditions (fixing step); (iii) heat the sample at constant rate (3°C/min) up to 70°C (recovering step). During the programming and recovering steps the specimens were maintained under a constant load of 0.001 N. In some experiments, thermal annealing was applied to the sample after the fixing step. Briefly, at the end of the programming step, the sample was quenched to -60°C, equilibrated at the annealing temperature (either 15°C or 10 ACS Paragon Plus Environment

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30°C) for 180 minutes and cooled down to -60°C. The recovering step was then performed as described above. The shape memory behavior was described in terms of strain fixity ratio (Rf), which quantifies the amount of the applied strain fixed at the end of the cooling step, and strain recovery ratio (Rr), representing the amount of applied strain recovered by the specimen during heating to 70°C, and thus the ability to recover its pristine strain. These parameters were calculated as follows:

ε  R f (%) =  unl  ×100 ε   appl 

(1)

ε −ε  Rr (%) =  appl rec  ×100  ε −ε   appl 0 

(2)

where εappl is the applied deformation (at the end of the programming step), εunl is the strain after load removal (at the end of the fixing step), εrec is the residual strain measured after the recovering step, and ε0 is the strain before deformation. Cell culture. NIH/3T3 fibroblast cells (ATCC CRL-1658; American Type Culture Collection, Rockville, MD, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) enriched with 10% fetal bovine serum, glutamine (2mM), penicillin (100U/mL) and streptomycin (100mg/mL) (Euroclone, Milan, Italy). Prior to cell experiments, the HMW-ABA electrospun samples were rinsed with PBS enriched with antibiotics (glutamine 6mM, penicillin 300U/mL, streptomycin 300mg/mL). For MTS assay, 1.5x105cells/well were seeded onto 1x1cm2 HMW-ABA electrospun scaffold in a 12 well plate. Culture plates were used as control. To observe cell morphology, 7.5x104cells/well were seeded onto materials and phalloidin staining has been performed. Viability assay (MTS assay). In order to evaluate cell viability cells were cultured on the HMWABA electrospun samples while TCP wells were used as a control. A [3-(4,5-dimethylthiazol-2-yl)11 ACS Paragon Plus Environment

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5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)- 2H-tetrazolium, inner salt (MTS) solution was added to culture medium without phenol red. After 3 h, culture medium was collected and the absorbance was read by UV–Vis spectroscopy (V-630 UV–Vis Spectrophotometer, Jasco, USA) at 490 nm. The absorbance was directly proportional to viable cells amount. The measures have been normalized by the area of culturing. Cell viability has been evaluated after 1 and 3 days of culture. Phalloidin staining. Cell morphology was observed using fluorescent staining. Briefly, at each time point (1 and 3 days of culture), cells were fixed in 4% formaldehyde water solution for 60 min at RT. After rinsing with PBS, phalloidin - tetramethylrhodamine (TRITC) conjugated (Sigma, Italy) was incubated for 45 min at 37°C in the dark. For nuclear staining, 4',6-diamidino-2phenylindole (DAPI) was used (Sigma, Italy). Cells were observed by fluorescent microscopy (Leica Microsystems DM2500) at 200X of magnifications.

RESULTS AND DISCUSSION Copolymer synthesis and characterization. In the current study, the molecular structure of the new HMW-ABA triblock copolymer was specifically designed to display the thermal and mechanical properties requested to obtain a material with shape memory performance in the physiological temperature range. In particular, the P(PAz60Seb40) block (B block) was chosen as the switching segment, and, based on results of previous work,49,50 it was synthesized with a chemical composition able to guarantee a crystal phase melting in the range of the physiological temperature. Poly(L-lactic acid), which melts at higher temperature than P(PAz60Seb40),51,52 was chosen as a hard segment to provide the physical net-point. The chemical structure of the B block, evaluated by 1H-NMR (Figure S1), was consistent with the expected structure and the actual composition was very close to the feed, i.e. 62.5% and 37.5% of PAz and PSeb co-units, respectively. 1H-NMR has been also employed for the determination of the 12 ACS Paragon Plus Environment

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molecular weight of the prepolymer, in accordance to the procedure previously reported, with minor modifications.53 The calculated value (Mn = 11200 g/mol) is comparable to that obtained by GPC (Mn = 12500 g/mol, PDI = 2.2). It might be worth highlighting that the prepolymer synthesis procedure has been optimized to achieve a quite high molecular weight, while preserving a high amount of OH-terminal groups, needed for the ring-opening of the lactide monomer. Accordingly, titration experiments demonstrated a very low concentration of carboxylic terminal groups (36± 6) x -3

10 mmolNaOH/g.

The HMW-ABA copolymer was obtained, as described in the Experimental Section and outlined in Scheme 1, by in situ ring opening polymerization of L-lactide, starting from P(PAz60PSeb40) prepolymer as initiator, and successive chain extension process through HDI addition, to increase triblock copolymer molecular weight. No unreacted HDI was detected by 1H-NMR NMR after the purification process. The 1H-NMR spectrum of HMW-ABA is reported in Figure 1, together with the resonance assignments. The HMW-ABA copolymer is composed for the 62.7 mol% of A and for the 36.1 mol% of B. Moreover, 1.2 mol% of HDI has been incorporated into the polymer. GPC experiments evidenced that a high molecular weight was achieved and a good control over the whole polymerization process was maintained (Mn = 40000 g/mol, PDI = 2.2).


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Figure 1.1H-NMR spectrum of HMW-ABA with resonance assignments.

Thermogravimetric analysis was carried out on the HMW-ABA copolymer, in order to evaluate its thermal stability, and on the plain B block for the sake of comparison. Thermal degradation occurred in two distinct weight loss steps (Figure S2) whose entity closely reflects copolymer composition, as calculated by 1H-NMR. The step at lower temperature (Tmax = 272°C) is attributed to the degradation of PLLA (A blocks), while that at higher T (Tmax = 416°C) to the decomposition of the B block, as confirmed by the TGA curve of the plain B block (Tmax = 417°C, Figure S2). Figure 2 shows the calorimetric curves of the synthesized HMW-ABA triblock copolymer and of the B block for the sake of comparison, whereas the corresponding calorimetric data are reported in Table 1. The triblock copolymer (Figure 2A) is semicrystalline and it is characterized by the thermal transitions of both the A and B blocks. The Tg at around -60°C, and the multi-peak melting endotherm in the temperature range 0-50°C, can be ascribed to the softening of the glassy phase and 14 ACS Paragon Plus Environment

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to the melting of the crystal phase of the B block, respectively, on the basis of the calorimetric scan of B block (Figure 2B). The high-temperature melting endotherm (Tm around 150°C) is associated to the melting of PLLA block crystal phase while the PLLA Tg, which is assumed to be in the range 30-50 °C, is not observable due to the melting endotherm of the B block. The lower cold crystallization enthalpy value (∆Hcc) compared to the melting enthalpy one (∆Hm) in the second heating scan of the HMW-ABA copolymer (Table 1), demonstrates that the PLLA block partially crystallize during quenching from the melt. The presence of two distinct crystalline phases in the HMW-ABA copolymer, attributed to the A and B blocks, was also confirmed by the WAXD pattern reported in Figure S3 that compares the diffractogram of the HMW-ABA copolymer with that of the B block and that of a semicrystalline PLLA (Figure S3). Indeed, the copolymer showed the reflection peak at around 16° typical of PLLA (A block), and the four reflection peaks at around 6° and in the range 18° - 25°, all of them present in the B block.


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Figure 2. DSC scans of (A) HMW-ABA and (B) B block (a: first scan; b: second scan after quench).

Table 1. Thermal characterization of the HMW-ABA copolymer and of the B block.

Tga (°C) HMW-ABA (1st scan) HMW-ABA (2nd scan) B block (1st scan) B block (2nd scan)

B block ∆Cp Tm a (J/g °C) (°C) a

∆Hma

b

(J/g)

Tcc (°C)

A block ∆Hccb Tm b (J/g) (°C)

∆Hmb (J/g)

-54

0.25

39

28

106

0.75

149

16

-56

0.25

13;29

19

86

12

150

17

-57

1.48

17;32;41

48

-

-

-

-

-57

1.64

17;30;41

47

-

-

-

-

Electrospun scaffold fabrication. The HMW-ABA copolymer was successfully processed to obtain electrospun scaffolds made of randomly arranged bead-free fibers with a mean diameter distribution of 1.1 ± 0.2µm (Figures 3A and B). After the electrospinning process the polymer still displayed the characteristic thermal properties of the pristine material (see DSC scan in Figure 3C): the low-melting crystal phase of the B block and the high-melting crystal phase of the A block. It is worth noting that the PLLA block is capable to partially crystallize during the electrospinning process, as demonstrated by the melting endotherm in Figure 3C and by the absence of a significant cold crystallization exotherm. However, it is widely accepted that PLLA is a slowly crystallizable polymer compared to many conventional thermoplastics54 and as-electrospun PLLA fibers are known to be completely amorphous just after the process.48 The capability of the PLLA block to crystallize in our polymeric system can be reasonably related to its low molecular weight (estimated to be 4000 g/mol) that determines a shift of PLLA ‘crystallization window’ to lower temperatures compared to the high molecular weight PLLA. This hypothesis is confirmed by both the not 16 ACS Paragon Plus Environment

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detectable PLLA Tg and the relatively low Tm (around 150°C) of the PLLA segment. The enhanced mobility of the PLLA segment in this particular polymeric architecture leads to a faster crystallization kinetics and, therefore, to the development of a crystal phase during the electrospinning process. This is of primary importance for the designed material, since the presence of PLLA crystal phase is necessary for the scaffold to exhibit shape memory properties.

Figure 3. (A) SEM micrograph, (B) fiber diameter distribution and (C) DSC (first scan) of electrospun HMW-ABA.


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Shape memory behaviour. A thermo-mechanical cycle, as described in the Experimental section, was used to characterize the shape memory properties of the electrospun copolymer. On the basis of the calorimetric results the cycle was planned by exploiting the B block crystal phase as molecular switch and the A block crystal phase as the physical network of the polymer chains. The material was (i) heated at 50°C, to completely melt the switching block and to allow relaxation of macromolecular orientation related to stretching and elongation of the polymer network in the electrospinning process; (ii) cooled to 30°C, i.e. below PLLA Tg, and (iii) deformed to confer the temporary shape that was subsequently fixed by cooling the sample at -60°. By applying the programming step at a T below PLLA Tg it is expected that the deformation was only applied to the switching phase (B block) component. Then, during the cooling, the applied strain was fixed thanks to the formation of B block crystal phase. To recover the permanent shape, and thus completing the thermomechanical cycle, the material was heated again above B block Tm (Figure 4A-B). Strain recovery as a function of temperature is reported in Figure 4C. The recovery of the permanent shape takes place starting from around -20°C (Figure 4C-b) reaching a value of around 10% at 20°C. The recovery becomes more evident in the range 20-30°C where the scaffold recovers about 90% of the initial strain (Figure 4C-a). Such recovery behavior, spanning a wide temperature range (from around -20°Cto around 30°C), can be ascribable both to the multi-peak melting endotherm of P(PAz60Seb40) crystals and to the broad melting region that starts well below RT (see DSC curve in Figure 3). Table 2 reports Rf and Rr calculated according to Equations (1) and (2), respectively. It is worth pointing out that at the end of the thermomechanical cycle, after the scaffold completely recovered its macroscopic permanent shape, the microscopic fibrous structure is maintained thanks to the presence of the high-melting phase of PLLA (Figure 4D).


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Figure 4. (A) Schematic representation of the mechanism of shape-memory for HMW-ABA; (B) 3D representation of the applied thermo-mechanical cycle; (C) strain recovery evolution over temperature in the range 0- 50°C (a) and from -60°C to 25°C (b); (D) SEM micrograph of the electrospun scaffold after the application of the thermo-mechanical cycle.


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Table 2. Applied strain (εapp), shape fixity ratio (Rf) and shape recovery ratio (Rr) of as-spun HMWABA and annealed electrospun HMW-ABA at 15°C and 30°C. sample HMW-ABA as-spun HMW-ABA annealed at 15°C HMW-ABA annealed at 30°C

εapp (%) 140 110 150

Rf (%) 99 98 99

Rr (%) 96 98 99

As reported above, the broad melting region of the B block results in a recovery of the permanent shape that starts at around -20°C (Figure 4C-b) and about 10% of the strain is recovered close to the RT range, thus entailing a partial loss of the fixed temporary shape when the material is used at RT. In order to overcome this limitation and optimize the shape memory behavior, different annealing treatments were applied to narrow the broad and multiple melting endotherm of the B block. As a preliminary investigation, Temperature Modulated DSC (TMDSC) was performed on electrospun HMW-ABA scaffold in order to identify the proper annealing temperature. In fact, TMDSC allows to separate the concomitant phenomena of cold crystallization and melting of the B block, which are overlapped in a conventional DSC measurement. The resulting calorimetric curves are shown in Figure 5A, where the non-reversing heat flow component displays a multi-peak crystallization occurring between Tg and Tm of the B block (Figure 5A-a). On the basis of TMDSC, the annealing was carried out by maintaining the sample mounted in the DMTA either at 15°C or 30°C for 3 h, after the programming and the fixing step, as described in the Experimental Section. To verify the effectiveness of the annealing, the samples were then analyzed through TMDSC in the temperature range of the switching segment melting, and the results are reported in Figure 5B together with the not annealed sample. As expected, the annealing had the effect of partially eliminating the low melting crystal phase, by increasing the amount of crystals that melt at T > Tannealing and by shifting the Tonset of the B block melting endotherm at higher temperature compared to the as-spun scaffold. 20 ACS Paragon Plus Environment

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The effect of the annealing treatment on the scaffold shape memory behavior, in terms of strain recovery as a function of temperature, is shown in Figure 5C. The curve of the as-spun sample (reported above in Figure 4C) is also shown for comparison. All samples display a recovery process in a T range that depends on the annealing treatment. In particular, after the annealing at 15°C the sample starts to recover the permanent shape at around 25°C and completes the recovery at 40°C. After the annealing treatment at 30°C, the strain recovery starts around 35°C and is completed at around 55°C. It is worth noticing that for both samples obtained after the two annealing treatments (at 15°C and at 30°C), the Rr and Rf values were excellent, with recovery indexes even better than those obtained for the as-spun HMW-ABA sample (Table 2). This ‘annealing-controlled’ shape memory behavior is strictly related to the structural perfection of the B block crystal phase, and hence to the corresponding melting transition, that is dependent on the polymer thermal history. In particular, the annealing treatment applied in this work allows the decrease of the amount of low melting crystals, by increasing the structural perfection of crystallites, moving the melting of the switching phase at higher temperatures (Figure 5B). Since the recovery of the permanent shape occurs when the switching phase gains enough mobility to release the strain energy stored during shape fixing, the annealing has the effect of stabilizing the material at RT and of shifting the recovery temperature at higher values. These results demonstrate that by applying a simple thermal treatment, the shape memory behavior and switching T of the HMW-ABA fibrous scaffold can be tuned without any change to the material composition. The obtained results suggest that a clever design of polymer microstructure and of thermal transitions might be a useful strategy to better exploit the potential of thermal responsive shape memory polymers.


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Figure 5. (A) TMDSC curves of electrospun HMW-ABA: (a) non reversing heat flow, (b) reversing heat flow, and (c) total heat flow. (B) TMDSC curves (total heat flow) of (a) not annealed sample, (b) sample annealed at 15°C, (c) sample annealed at 30°C. (C) Strain recovery evolution over temperature in the range 0-60°C for samples: (a) not annealed, (b) annealed at 15°C and (c) annealed at 30°C.

Biocompatibility of the new shape memory scaffolds. In order to evaluate the biocompatibility of the new shape memory scaffolds, at 1 and 3 days NIH/3T3 fibroblast cells viability has been evaluated by MTS assay (Figure 6). Cells showed an optimal viability when seeded on the electrospun HMW-ABA scaffold, that increased with time. After 1 and 3 days of culture, phallodin and DAPI were used to observe cell morphology (Figure 6). Already after 1 day of culture, 22 ACS Paragon Plus Environment

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NIH/3T3 fibroblasts were well spread and showing the characteristic spindle morphology (elongated cytoplasm). Unaltered cell morphology was maintained and cell proliferation was adequate compared to the control. These preliminary results are promising in view of the application of the shape memory electrospun devices as functional tissue engineering scaffolds, as recently demonstrated by Bao et al..35 Such scaffolds combine the biomimicking characteristic of the fibrous structure with the shape memory effects to allow minimally invasive surgical implantation and to achieve an enhanced efficacy in tissue repair and regeneration by controlling the cell behavior through mechanicalbiological stimuli.

Figure 6.(A) Phalloidin staining for cell morphology and (B) cell viability of NIH/3T3 seeded on HMW-ABA scaffold. Data are statistically significant with respect to control. * indicates p≤0.05.


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CONCLUSIONS In this work a new linear PLLA-based triblock copolymer was proposed to fabricate a scaffold for tissue engineering applications, displaying a thermally induced shape-memory behavior around physiologically relevant temperature. The copolymer microstructure was designed to provide a low melting crystal phase acting as switching segment, with the capability to promptly fix the temporary shape of the SMP, and a high melting crystal phase acting as physical network. Remarkably, the monomers adopted for the copolymer synthesis, with the exception of a small amount of HDI used as chain extender, are characterized by a 100% biobased character, as being lactic acid, 1,3propanediol, azelaic and sebacic acids. The switching segment central block, i.e. poly(propylene azelate-co-propylene sebacate) (60/40) random copolymer, is characterized by a chemical composition which guarantees a crystal phase melting in the range of the physiological temperature; the PLLA sequences provide the physical network. The copolymer was suitable to be processed through electrospinning to obtain a microporous scaffold made of randomly arranged bead-free fibers. The shape memory properties of the scaffold were characterized through a thermo-mechanical cycle programmed on the basis of a throughout thermal characterization of the material. In particular, it was shown for the first time that a thermal annealing treatment, whose aim was to narrow the broad and multiple melting endotherm of the switching phase by increasing crystal phase perfection and melting temperature, was effective in controlling the shape-memory trigger temperature interval. It is worth pointing out that at the end of the thermomechanical cycles, the scaffold completely recovered its macroscopic permanent shape, while well maintaining its microscopic fibrous structure. In conclusion, in this work we demonstrated that, by controlling the melting and recrystallization processes, it is possible to shift the shape recovery temperature range without the need of varying the copolymer’s chemical structure. Hence, the annealing approach reported in this work turned out 24 ACS Paragon Plus Environment

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to be a smart tool to obtain a shape memory material with tunable recovery behavior. More generally, the approach here adopted can be considered a useful strategy to tailor the shape recovery temperature of a given polymer, thus providing a simple, yet powerful way of ‘ad hoc’ programming the same material for different applications requiring different shape recovery temperatures. Biocompatibity of the proposed scaffold was demonstrated by using NIH/3T3 fibroblast cells. The attractive shape-memory behavior of the biomimetic microporous scaffold investigated in this work, combined with its biocompatibility make it suited for tissue regeneration, to allow minimally invasive surgical implantation and control of cell behavior through mechanical-biological stimuli.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. The following data are reported:1H-NMR spectrum of B; thermogravimetric curves of B and HMW-ABA; WAXD patterns of the HMW-ABA copolymer, of the B block and of PLLA. AUTHOR INFORMATION Corresponding Author * Email: [email protected] Tel.+39-051-209 9577; Fax: +39-051-209 9456 Present Addresses †Department of Chemical Science and Technology, University of Roma “Tor Vergata”, via della Ricerca Scientifica 1, 00133, Roma, Italy. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. 25 ACS Paragon Plus Environment

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Notes The authors declare no competing financial interest. ACKNOWLEDGMENT The Authors acknowledge the Italian Ministry of University and Research. MLF acknowledges the support of FP7 COST Action MP1206 “Electrospun Nanofibres for bioinspired composite materials and innovative industrial applications”.

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Table of Contents Graphic


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Figure 1.1H-NMR spectrum of HMW-ABA with resonance assignments. 102x72mm (300 x 300 DPI)

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Figure 2. DSC scans of (A) HMW-ABA and (B) B block (a: first scan; b: second scan after quench). 80x114mm (300 x 300 DPI)


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Figure 3. (A) SEM micrograph, (B) fiber diameter distribution and (C) DSC (first scan) of electrospun HMWABA. 80x161mm (300 x 300 DPI)


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Biomacromolecules

Figure 4. (A) Schematic representation of the mechanism of shape-memory for HMW-ABA; (B) 3D representation of the applied thermo-mechanical cycle; (C) strain recovery evolution over temperature in the range 0- 50°C (a) and from -60°C to 25°C (b); (D) SEM micrograph of the electrospun scaffold after the application of the thermo-mechanical cycle. 170x207mm (300 x 300 DPI)


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Figure 5. (A) TMDSC curves of electrospun HMW-ABA: (a) non reversing heat flow, (b) reversing heat flow, and (c) total heat flow. (B) TMDSC curves (total heat flow) of (a) not annealed sample, (b) sample annealed at 15°C, (c) sample annealed at 30°C. (C) Strain recovery evolution over temperature in the range 0-60°C for samples: (a) not annealed, (b) annealed at 15°C and (c) annealed at 30°C. 80x135mm (300 x 300 DPI)


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Figure 6.(A) Phalloidin staining for cell morphology and (B) cell viability of NIH/3T3 seeded on HMW-ABA scaffold. Data are statistically significant with respect to control. * indicates p≤0.05. 80x107mm (300 x 300 DPI)


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Scheme 1. Reaction scheme for the synthesis of the HMW-ABA copolymer. 174x118mm (300 x 300 DPI)


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Thermal Annealing to Modulate the Shape Memory ... - ACS Publications

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