Amphiphilic Nucleating Agents to Enhance Calcium Phosphate

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Amphiphilic Nucleating Agents to Enhance Calcium Phosphate Growth on Polymeric Surfaces Laura C. E. da Silva,† Bruna A. Más,‡ Eliana A. R. Duek,‡ Richard Landers,§ Celso A. Bertran,† and Maria C. Gonçalves*,† †

Institute of Chemistry, University of Campinas, P.O. Box 6154, Campinas 13083-970, Brazil Faculty of Medical Sciences, Pontifical Catholic University of São Paulo − PUC-SP, Sorocaba 18030-095, Brazil § Laboratory of Surface Physics, Department of Applied Physics, “Gleb Wataghin” Institute of Physics, University of Campinas, P.O. Box 6165, Campinas 13083-859, Brazil ‡

S Supporting Information *

ABSTRACT: Poly(ε-caprolactone) (PCL) is an aliphatic polyester widely explored in the preparation of guided bone regeneration (GBR) membranes because of its interesting mechanical properties and biodegradability. However, PCL high hydrophobicity often impairs cell adhesion and proliferation as well as calcium phosphate growth, all of which are crucial to achieving suitable bone−tissue integration. In this work, aimed at achieving less-hydrophobic surfaces, amphiphilic molecules were added at low concentrations to the polymeric dope solutions that generated the GBR membranes. During membrane formation, these molecules migrate to the solution/air interface in such a way that, upon liquid−solid phase transition, the negatively charged heads are exposed while the apolar tails are anchored to the polymer bulk. As a consequence, these molecules became nucleating agents for subsequent calcium phosphate growth using an alternating soaking process. Herein, PCL porous membranes containing different amphiphilic molecules, such as stearic acid and bis(2-ethylhexyl) phosphate, were investigated. This new, simple, and atoxic method to superficially treat polymeric membranes could be extended to a wide range of polymers and applications.

1. INTRODUCTION Calcium phosphate (CaP) coatings have been widely explored as a means of enhancing ostegenesis in implantable devices. The mechanism by which it is achieved is not yet fully understood. However, Surmenev et al.,1 in their review article, summarized the main aspects of implant-tissue integration promoted by CaP coatings. Briefly, upon implantation, coated devices suffer partial CaP solubilization, which increases ionic activity at the bone/implant interface mediated by the physiological fluids present. Calcium ions now present at the interface induce protein adsorption, which signals cell adhesion and proliferation. Finally, CaP reprecipitation occurs simultaneously with cell spreading and tissue ingrowth. This strategy has proven to be suitable for enhancing biocompatibility and promoting osteogenesis in metallic,2 ceramic,3 and polymeric4 biomaterials. However, coating characteristics such as the CaP morphology,5 crystal structure,6 thickness,7 porosity,8 and topography4 are limiting factors in assuring biocompatibility and osteogenesis and may be controlled by the CaP deposition methods selected. Recently, in a thorough review, Dorozhkin7 listed all available techniques to achieve CaP coatings. In polymeric systems, wet techniques are the most widely explored because of substrate © XXXX American Chemical Society

limitations on high pressure and high temperature, both of which are needed for the thermal spraying and vapor deposition methods. Nevertheless, despite the deposition method chosen, the main challenge in achieving well-adhered CaP coatings in polymeric devices is their hydrophobic surface.9 For this reason, in the vast majority of cases, it is necessary to perform a pretreatment on the device surface prior to coating and/or to endure low CaP nucleation rates.7 Pretreatments may be divided into three categories: mechanical treatments, such as sanding;10 physical treatments, such as plasma;11 and chemical treatments, such as polymer grafting12 or hydrolysis.13 Chemical treatments are the most widely studied because of their efficiency and cost effectiveness; however, these chemical pretreatments frequently generate high amounts of residue and require toxic reagents, which may not be completely removed.7,1 In their work, Tanahashi et al.14 investigated the effect of surface charge on CaP deposition using a model surface consisting of modified alkenethiols bound to gold-coated Received: December 20, 2016 Revised: March 1, 2017 Published: March 27, 2017 A

DOI: 10.1021/acs.langmuir.6b04562 Langmuir XXXX, XXX, XXX−XXX

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susceptible to hydrolysis. The latter is the main reason that so many authors choose PCL as a substrate. Commonly, PCL is used in the form of thin films,19 electrospun membranes,24 and prototyped 3D25 devices that are subjected to alkali hydrolysis to produce carboxylate groups that are able to bind to CaP nuclei. These carboxylate groups are produced as a consequence of surface degradation through ester bond cleavage.17 As an alternative, some authors have blended PCL with natural hydrophilic polymers such as gelatin26 or silk fibroin.27 However, this blending approach has proven to increase the PCL degradation rates even further because of increased water uptake. In terms of device preparation, most studies use the eletrospinning process to produce membranes.28 Membranes are highly attractive in dentistry because they are crucial to the guided bone regeneration (GBR) therapy used in periodontal surgery. Recently, our group proposed a highly reproducible and cost-effective alternative to preparing GBR membranes.29 This method, based on vapor-induced phase separation (VIPS), produces asymmetrically porous membranes from polymeric dope solutions, allowing high productivity, and is easily adaptable to any polymer of interest.30−33,29 Herein, PCL membranes were prepared through VIPS and coated with calcium phosphate. During membrane formation, amphiphilic molecules, added to the PCL solution, can migrate to the solution/air interface in such a way that, upon liquid− solid phase transition, the negatively charged heads are exposed while the apolar tails are anchored at the polymer bulk. As a consequence, these molecules became nucleating agents to subsequent calcium phosphate growth through an alternating soaking process. In this work, PCL porous membranes containing different amphiphilic molecules, such as stearic acid and bis(2-ethylhexyl) phosphate, were investigated. This new, simple, and atoxic method to superficially treat polymeric membranes could be extended to a wide range of polymers and applications.

quartz. The authors showed that negatively charged surfaces (provided by phosphate or carboxylate groups attached at the alkenethiol free end) are more efficient than positively charged surfaces (provided by amine groups attached at the alkenethiol free end) in promoting CaP nucleation. In addition, this work showed that neutral surfaces (provided by methyl groups chemically attached at the alkenethiol free end) are unable to produce CaP nucleation in the 7 day period investigated. The authors attribute this phenomenon to the fact that the first step toward calcium phosphate nucleation and growth is calcium ion adsorption, which is favored by negative charges due to ionic bonding. After Tanahashi’s work, several authors tried, through graftization,15 silanization,16 oxidation,17 and hydrolysis13 to promote negatively charged surfaces on innumerable polymeric devices. It is now widely accepted that alkali hydrolysis18 is the most effective route to achieving this goal. For this reason, there are several studies available on the subject.13,18−20 Low CaP nucleation rates are another drawback for CaP coatings in implantable devices. Usually it takes at least 6 h,18 but most commonly a couple of days,21 to achieve stable CaP stable nuclei even on negatively charged surfaces. Oyane et al.,18 Tanahashi et al.,14 and others have already shown that the kind of functional group that promotes charge is a significant factor to consider in order to increase the CaP nucleation rate. Nonetheless, most authors have been focusing on manipulating the calcium and phosphate source instead.22 This approach is based on the assumption that, upon increasing the surface wettability, calcium ions are more easily adsorbed at the surface. However, Mavis et al.8 have shown that the calcium and phosphate sources have a great influence on the CaP structure and crystallinity. Aiming to solve this issue, Taguchi et al.23 were the first to propose the addition of a new step, the alternating soaking, to this process. The alternating soaking, or alternate dipping, is to be performed in between surface pretreatment and immersion in simulated body fluid (the most common calcium and phosphate source). It consists of dipping the intended device alternately in calcium-rich and phosphate-rich solutions, for a given number of cycles, in order to achieve a high calcium ion concentration at the surface and consequently promote CaP nucleation. This method increases the CaP nucleation rate, allowing stable CaP nuclei to be achieved in less than 1 min. The number of cycles used is usually low, less than 10 cycles. Nonetheless, some authors have used up to 150 cycles to attain complete coverage of the device surface. The association of Taguchi’s23 alternate soaking with Tanahashi’s14 findings on negative charges is, until now and to the best of our knowledge, the simplest and most effective way of producing CaP coatings on polymeric devices. Nevertheless, a cost-effective, atoxic, and efficient method of obtaining highly available negative charges at the polymer surfaces is yet to be reported. Notwithstanding the significant amount of work done in this field of research, little attention has been given to the polymeric substrate characteristics and even less attention has been given to device preparation. Most authors do not correlate the particular polymer characteristics, such as chemical structure, biocompatibility, and biodegradability, to the results found in their work. As a consequence, few studies consider the device preparation and performance as a whole. The most widely investigated polymer in this field of research is poly(ε-caprolactone) (PCL). PCL is a flexible, highly crystalline, biodegradable aliphatic polyester that is highly

2. EXPERIMENTAL SECTION 2.1. Materials. Poly(ε-caprolactone) (PCL) with a 150 kg mol−1 weight-average molecular weight (Mw) was supplied by Scientific Polymer (Ontario, Canada, catalog no. 1034). Tetrahydrofuran (THF), stearic acid (C18H36O2), calcium nitrate (Ca(NO3)2), and diammonium hydrogen phosphate ((NH4)2HPO4) were supplied by LabSynth (São Paulo, Brazil). Acrydin orange (C12H19N3) and bis(2ethylhexyl) phosphate (C16H34PO4) were supplied by Sigma-Aldrich (New York, USA). 2.2. Membrane Preparation. PCL porous membranes were prepared as described in previous work.29 Briefly, a dope solution containing 11.0 wt % PCL, 10.0 wt % water, and 0.2 wt % of the desired CaP nucleating agent, using THF as a solvent, was spread between Ni−Cr wires set on a glass plate and placed in an acrylic chamber with relative humidity above 75% for 5 h. Afterward, the membranes were removed and dried at room temperature for 24 h. The molecular structures of the nucleating agents used in this work are presented in Chart 1. 2.3. Calcium Phosphate Growth. Calcium phosphate particle growth was inspired by the work of Serizawa et al.15 and optimized in a 24−1 with a central point experimental design in which the effect of temperature, number of cycles, immersion time, and Ca2+/PO4−3 concentration for calcium phosphate nucleation and growth were simultaneously investigated. Supporting Information (S1−S3) shows detailed information on the experimental design. The optimized procedure, shown in Scheme 1, consists of dipping the membrane alternately in a 400 mmol L−1 Ca(NO3)2 solution and in a 240 mmol L−1 (NH4)2HPO4 solution for 30 s each. Between solutions, the B


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energies, the C 1s 284.6 eV binding energy, attributed to alkyl groups, was used as a reference. Cytocompatibility measurements through the MTT assay were performed as described elsewere.29 Briefly, primary osteoblast cells were extracted from calvaria fragments of 20-day-old Wilstar rats and cultured at 37 °C in a 5% CO2 humid atmosphere in DMEMsupplemented culture medium. Circularly shaped sections (5 mm each) were cut from each membrane, sterilized, and placed on 96-well culture plates. Osteoblasts at passages 6 and 7 were seeded on top of the membranes at a 1 × 103 cells mL concentration, and MTT formation was evaluated using the Mosmann35 protocol at 1, 5, and 12 days of culture. Hydrolytic degradation experiments were performed according to ASTM F1635-11. Briefly, four sections of approximately 6 mg and 0.25 cm2 were cut from each membrane, placed separately in vials containing 12.5 g of deionized water, and kept at 37 °C. After 30 days, these sections were weighed using an AD-6 (PerkinElmer) microbalance, and their molar masses were evaluated by a VE2001 GPCmax (Viscotek) gel permeation chromatograph (GPC) equipped with a 2500 UV detector and OMNISec software. The results presented are an average of four sections.

Chart 1. Nucleating Agents’ Chemical Structures

membranes were immersed in deionized water for 10 s and dried on filter paper. In all, 10 soaking cycles were performed. Afterward, the membranes were dried at room temperature for 24 h. 2.4. Characterization. Contact angle measurements were performed in a Theta Lite Optical (Attesion) tensiometer using a 2.5 μL drop of deionized water. The drop shape was recorded for 90 s, and the measurements were taken from the linear coefficient obtained by the linear fitting of each curve in the 30 to 90 s range. In all, 10 measurements were taken from each sample at the bottom surface. The morphologies of the samples were investigated using a TCS XP5 (Leica) confocal laser scanning microscope operating with a 488 nm laser and equipped with a photomultiplier, operating in the 520 to 540 nm wavelength range. Membranes were previously dipped 20 times in a 1 × 10−5 mol L−1 acrydin orange solution for 30 s each. Between dippings, the membranes were washed in deionized water and dried on filter paper. This process was performed to simulate the alternating soaking process described in the previous section. Differential scanning calorimetry (DSC) was performed in a Q100 (TA Instruments) equipment by using approximately 5 mg samples. Briefly, measurements were performed at 20 °C min−1 from 10 to 100 °C at the first heating, from 100 °C to −90 °C at cooling, and from −90 to 100 °C at the second heating. The degree of crystallinity was calculated from the melt peaks during the second heating using the equation described by Liu et al.34 Membrane surfaces were carbon and gold−palladium sputter coated in a Bal-Tec MD20 (Balzers) sputter coater and investigated in a JSM6340 (JEOL) field emission scanning electron microscope (SEM) operating at an accelerating voltage of 3 kV. Thermogravimetric analyses (TGA) were obtained in a 2950 (TA Instruments) thermobalance over a 25 to 800 °C temperature range and at a 10 °C min−1 heating rate under an argon atmosphere. Approximately 10 mg samples were used. Attenuated total reflectance infrared (ATR-IR) spectra were obtained on a Cary 630 (Agilent) spectrophotometer over a 4000 cm−1 to 400 cm−1 wavenumber range using 64 scans and 4 cm−1 resolution. X-ray photoelectron spectroscopy (XPS) was performed using a VSWHA-100 spherical analyzer with an aluminum anode (Al Kα 1456.6 eV) at pressures below 2 × 10−6 Pa. To correct the binding

3. RESULTS AND DISCUSSION In this work, asymmetrically porous PCL membranes with a 52 vol % approximate porosity, prepared by VIPS, were used as substrates to investigate the effect of the nucleating agents on calcium phosphate (CaP) formation by alternating soaking. The membrane general morphology obtained by this preparation method is shown in previous work29 and is illustrated in Supporting Information S4. On the basis of the work of Tanahashi et al.,14 which verified that carboxylate and phosphate groups are the most efficient functional groups, stearic acid (PCL-A) and bis(2-ethylhexyl) phosphate (PCL-E) were chosen as nucleating agents. The idea behind adding a small amount of an amphiphilic nucleating agent to a polymeric membrane is to promote charged, or chargeable, sites at the surface. These charged sites are useful for increasing the water wettability and provide binding sites for calcium ions, as suggested by Watanabe et al.36 Nevertheless, it is fair to assume that above a limiting concentration, excess nucleating agent molecules at the polymer bulk could act as plasticizers, as nucleating agents for polymer crystallization, or even as polymer degradation promoters. Scheme 2 shows the membrane preparation process and is key to elucidating the methodology proposed herein. Initially, a dope solution containing polymer, nucleating agent, solvent, and water is cast on a glass plate and exposed to a humid atmosphere. Over time, the solvent evaporates and liquid− liquid phase separation occurs. As liquid−liquid phase

Scheme 1. Demonstration of One Alternating Soaking Cycle for Calcium Phosphate Growtha

Schematic representation of one soaking cycle, which consists of dipping the membrane for 30 s in a 400 mmol L−1 calcium nitrate solution (1), followed by 10 s in deionized water (2) and filter paper drying. Then, the membrane was dipped for 30 s in a 120 mmol L−1 diammonium hydrogen phosphate solution (3), followed by 10 s in deionized water (4) and filter paper drying.

a

C


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(containing bis(2-ethyhexyl)phosphate as nucleating agent) showed 78 ± 1 and 78 ± 2° water contact angles, respectively. Even though the nominal value difference does not seem significant, over a period of 90 s the PCL water contact angle showed a decrease of 5.1 ± 0.5°, whereas those of PCL-A and PCL-E showed a decrease of 8 ± 1 and 10 ± 1°, respectively. These results indicate that the negative charges promoted by the nucleating agents were efficient at increasing the polymer wettability due to the increase in the hydrophilic character of the membrane surface. To verify the availability of the negative charges as binding sites for calcium ions and also as a proof of concept, PCL and PCL-A were acrydin orange-stained and imaged. Acrydin orange is a widely used dye that is positively charged in aqueous media. Confocal laser scanning microscopy (CLSM) was used to verify the dye uptake on the top membrane surface because of the negative charges promoted by the nucleating agent at the surface. In CLSM, brightness is dependent on the dye concentration. The PCL image (data not shown) is dark, and surface features such as porosity are not clearly distinguished. In contrast, PCL-A (Figure 1b) is significantly brighter, and the membrane characteristic porosity is clearly distinguished. This is an indication that PCL-A has increased dye uptake as a result of the negative charges uniformly distributed on the membrane surface. As mentioned earlier, above a limiting concentration, the nucleating agent excess molecules could cause changes in the polymer thermal properties due to plasticization, polymer nucleation, or even polymer degradation. Therefore, to determine if the nucleating agent concentrations (0.2 wt %) were not above that limit, DSC measurements (Table 1) and X-

Scheme 2. Demonstration of Membrane Formation from the Dope Solution Containing a Nucleating Agentb

b

A polymeric solution containing a dispersed nucleating agent is exposed to a humid atmosphere above the dew point. Because of solvent evaporation and water uptake, the solution undergoes liquid− liquid demixing, during which the nucleating agent migrates to the interface. Nucleating agents are amphiphilic molecules. Therefore, their polar ends turn toward the water-rich phase. Afterward, a liquid− solid transition takes place, anchoring the nucleating agent at a position in which its polar groups are exposed to air. These groups, during the alternating soaking, become anions that are then used to nucleate calcium phosphate growth.

separation occurs, the nucleating agent, which is an amphiphilic molecule, migrates to the interface. This migration occurs in such a way that the nucleating agent polar heads (carboxylate in PCL-A or phosphate in PCL-E) are in contact with the waterrich phase, which generates pores, whereas the nucleating agent apolar tails are located in the polymer-rich phase, which generates the membrane bulk. The polymer-rich phase, at a given moment, undergoes a liquid−solid phase transition during which the nucleating agent hydrophobic tails entangle with the polymeric chains. This entanglement anchors the negative charges provided by the nucleating agent polar heads at the membrane surface. To verify that the nucleating agent was able to produce negative charges at the polymer surface, water contact angle measurements of the membrane bottom surfaces were taken (Figure 1a). The membrane bottom surface, which was formed in contact with the glass plate, was chosen for these measurements because of the lower roughness compared to that of the top surface (Supporting Information S4). PCL showed an 80 ± 2° average contact angle, and PCL-A (containing stearic acid as a nucleating agent) and PCL-E

Table 1. Differential Scanning Calorimetry Results for the Membranes PCL PCL-A PCL-E

Tonset (°C)a

Tc (°C)a

Tg (°C)b

Tm (°C)b

X (%)b

29 28 29

20 22 22

−61 −61 −61

56 57 57

52 53 57

a

Results obtained from cooling: Tonset is the temperature at the beginning of crystallization, and Tc is the crystallization temperature. b Results obtained from the second heating: Tg is the glass-transition temperature, Tf is the melt temperature, and X is the degree of crystallization.

Figure 1. Contact angle measurements of PCL, PCL-A, and PCL-E untreated membranes (a) and confocal laser scanning micrograph of an acrydin orange-stained PCL-A untreated membrane (b). D


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Figure 2. Scanning electron micrographs of the PCL-A top surface showing pores (a) and CaP nuclei inside a pore (b).

Figure 3. Scanning electron micrographs of the untreated PCL membrane top surface (a) and calcium phosphate particles grown on the top surfaces of PCL- (b), PCL-A- (c), and PCL-E-treated (d) membranes.The inset scale bar is 5 μm.

ray diffraction (data not shown) were performed. Table 1 shows a summary of the DSC results. No significant changes in the glass transition, melt and crystallization temperatures or the degree of crystallinity were observed in the presence of both nucleating agents. As a consequence, it is fair to assume that stearic acid and bis(2-ethylhexyl) phosphate did not inpart any significant changes to the polymer thermal properties. Aiming to initially access stearic acid’s ability to produce stable CaP nuclei on the membrane surface, the first alternating soaking cycle was evaluated by SEM. In the neat PCL membrane (data not shown), with one soaking cycle alone, no CaP nuclei were observed. In contrast, in PCL-A CaP nuclei

are preferentially located in some regions (Figure 2a). (Figure 2b) shows an area of uniformly dispersed CaP nuclei. Two factors must be considered in explaining why the CaP nuclei are located in some regions and not in others. The first factor is the top surface morphology. The geometry of the large pores hinders aqueous solution access during the short time framework of one soaking cycle. The second factor deals with the nucleating agent location. It is quite probable that after membrane preparation not all nucleating agent molecules are located and exposed at the surface. Therefore, when the membrane is immersed in the aqueous solutions, such as the calcium and phosphate precursors, the nucleating agent molecules on the surface surroundings might diffuse to the E


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Figure 4. Thermogravimetric analysis (a) and attenuated total reflectance infrared spectra (b) of PCL-, PCL-A-, and PCL-E-treated membranes.

Table 2. X-ray Photoelectron Spectroscopy Results of PCL Membranes after Alternating Soaking Cycles binding energy (eV) C (1s)

sample PCL PCL-A PCL-E

284.6 284.6 284.6

286.1 286.6

atomic ratio

O (1s) 288.5 288.6 288.6

532.2

533.5 533.5

531.8 532.3

Ca (2p)

P (2p)

Ca/P

C/O

O/Ca

347.3 347.3 347.0

133.6 133.5 133.8

2.0 0.6 0.2

2.8 1.8 3.0

5.3

though PCL-E showed a significant decrease in degradation temperature, it still degradated in a single thermal degradation event, which is an indication that the PCL degradation mechanism remained unchanged. The attenuated total reflectance infrared (ATR-IR) spectra of the membrane’s bottom surface was taken before and after the alternating soaking treatment (Figure 4b). PCL characteristic bands at 2943 and 2856 cm−1 are associated with aliphatic C− H bonds; at 1155 cm−1, they are associated with C−O stretching; and at 1720 cm−1, they are associated with carbonyl groups, which are present in all of the samples.26 Neither stearic acid nor bis(2-ethylhexyl) phosphate was detected, probably because of the low concentration. After the alternating soaking treatment, PCL spectrum remained unchanged, also because of the extremely low CaP concentration. In contrast, in both PCLA and PCL-E, the polymer characteristic band intensities (at 1155, 2943, and 2856 cm−1) were significantly reduced after the alternating soaking treatment. This phenomenon is due to a decrease in PCL concentration at the outermost layer of the membrane. In the case of PCL-A, the PCL diminished concentration is associated with the rise in phosphate characteristic bands at 560 and 1030 cm−1.27 In the case of PCL-E, on the other hand, no phosphate characteristic bands were found. To further investigate the surface composition of PCL membranes after the alternating soaking treatment, X-ray photoelectron spectroscopy (XPS) was carried out, and the main findings are presented in Table 2. C 1s binding energies describe the presence of CH2−CH2 (284.6 eV), C−O (286.1 eV), and CO (288.5 eV) bonds, all of which are clearly attributed to the polymeric matrix. PCL did not show C−O bonds, and 94% of the C atoms analyzed by XPS for this sample refers to the CH2−CH2 bond. In PCL-A and PCL-E, 67 and 89% of the C atoms, respectively, refer to the CH2−CH2 bond. In both samples, the C−O bond is present. Oyane et at.17 correlated the increase in the C−O bond concentration and the decrease in the CH2−CH2 bond concentration to the cleavage of the polymer ester bonds and, consequently, to

surface, which would generate an increase in the nucleating agent concentration at the polymer surface over time. The initial heterogeneity in the CaP nuclei formation was totally overcome after 10 soaking cycles. However, it is important to stress at this point that the alternating soaking methodology is meant only to initiate the CaP deposition, not to thicken the cap coating. This is important in order to promote implant-tissue integration.15 Figure 3a shows the scanning electron micrographs of the PCL membrane prior to the alternating soaking cycles, and Figure 3b−d shows SEM images of the top surface of the membranes after 10 alternating soaking cycles. As can be seen, after the alternating soaking cycles, even neat PCL (Figure 3b) shows few CaP deposits. This is due to polymer hydrolysis at the surface, as described by Choong et al.19 Because the alternating soaking solutions are neutral, the extent of hydrolysis is low, so CaP deposits are sparse and the particles formed are irregular with a wide range of shapes and sizes. CaP particles in PCL-A (Figure 3c), conversely, mainly present spherical CaP on both surfaces indistinctly and, to some extent, penetrate the membrane bulk (Supporting Information S5). As shown above, CaP coverage on PCL-E (Figure 3d) is not as vast as on PCL-A; however, it is significantly more present than on neat PCL. In PCL-E, particles are larger and in the form of platelets. The distinct morphologies found in the presence of the different nucleating agents are direct evidence of the influence of the nucleating agent on CaP nucleation and growth. As suggested by Madurankatam,22 these distinct CaP morphologies may be a result of a distinct CaP chemical structure. However, because of the small amount of CaP deposited (below 2 wt % in all cases), the CaP chemical structure of each membrane was not possible to be determined. The CaP weight percentage was determined by the inorganic fraction verified by thermogravimetric analysis (Figure 4a) at 700 °C. TGA also shows PCL thermal degradation behavior. Stearic acid did not interfere with PCL degradation. Bis(2ethylhexyl) phosphate, on the other hand, decreased the PCL onset degradation temperature by 70 °C. Interestingly, even F


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absorbance to the control. This means that all samples were equally cytocompatible. On the fifth day, PCL absorbance was well above that of the control, whereas PCL-A was below and PCL-E was equal to the control. PCL absorbance dropped after 12 days, falling well below the control. Roach et al. reports the behavior of rapid proliferation followed by cell death as being due to the confluence of cells38 and usually pointing to poor device−tissue integration. However, statistical analysis showed that after 12 days, PCL-A and PCL-E were equal to the neat PCL reference sample because equilibrium was reached. It is worth mentioning that the delayed cell viability found in PCL-A and PCL-E samples is due to CaP dissolution that increased the acidity in the polymer surroundings, whereas the high error in PCL-E is due to the fact that PCL-E does not show complete CaP coverage.1 This result showed that both nucleating agents were able to generate cytocompatible membranes that were able to sustain viable cells for a 12-day period. In terms of hydrolytic degradation (Figure 6), the literature reports that PCL has a surface erosion degradation mechanism,

surface degradation. In this case, surface degradation is due to hydrolysis during the alternating soaking. In the PCL control sample, two O 1s binding energies may be found, 533.5 and 532.2 eV, and are attributed to the polymeric matrix CO and C−O− bonds, respectively. C−O− bonds correspond to cleaved PCL ester bonds.17 Therefore, it is fair to assume that the O atoms found in the PCL sample came from both degraded and undegraded polymer chains. In PCL-E, a single O 1s binding energy of 532.3 eV is verified and is attributed to C−O− bonds, which shows that the PCL-E surface is mainly composed of degraded polymer chains. This result is in agreement with the water contact angle measurements because PCL-E showed the highest wettability. Finally, in the case of PCL-A, not only there was no evidence of C−O− bonds (532.2 eV) but also 74% of the O atoms present may be attributed to phosphate groups thorough the O 1s binding energy of 531.8 eV. Moreover, approximately 7.5% of the atoms in the PCL-A outermost surface are either Ca or P, whereas in PCL and PCL-E, Ca and P combined correspond to less than 1.5% of the atoms. Also, the C/O atomic ratio in PCL-A is much lower than the polymeric matrix C/O theoretical atomic ratio (3.0). All of this evidence confirms the SEM (Figure 3) results: the PCL-A surface is mainly made up of CaP, whereas PCL and PCL-E surfaces are mainly made up of degraded and/or undegraded polymer chains. XPS measurements are also useful in determining the CaP structure through the analysis of Ca/P and O/Ca atomic ratios.37 Because of the fact that neither PCL nor PCL-E showed phosphate-related O atoms, the O/Ca atomic ratio of these samples could not be attained. On the basis of the available literature,37,7,1 the Ca/P atomic ratio found for the PCL sample suggests that the CaP structure in this sample is associated with amorphous calcium phosphate (ACP). The Ca/ P and O/Ca atomic ratios found in PCL-A suggest that the CaP structure is related to monocalcium phosphate monohydrate (MCPD). The Ca/P ratio found for PCL-E does not correspond to any known CaP structure. Figure 5 shows the cytocompatibility of the treated membranes evaluated in a 1, 5 and 12-day MTT assays. This experiment is used to quantitatively assess cell viability on a device surface and compare it to a control group, which in this case consisted of the polystyrene tissue culture plates and neat PCL. Initially, on the first day, all samples showed equal

Figure 6. Thirty day hydrolytic degradation assay of PCL-A and PCLE membranes after the alternating soaking cycles using neat PCL as a reference. Bars represent the sample molar mass before and after the 30-day hydrolytic degradation assay, and the line shows the sample weight loss after the 30-day hydrolytic degradation assay.

which means that during degradation the device weight loss is favored over the polymer molar mass decrease.39 Both nucleating agents preserved this degradation mechanism, even though the PCL-E initial molar mass was significantly lower, because of the fact that in a 30 day period the polymer molar masses remained unchanged and all samples showed similar weight losses. PCL-A showed a slight increase in its weight loss percentage due to CaP dissolution, as also verified in the MTT assay. One possible explanation for the fact that PCL-E showed a significantly lower starting molar mass, even though it remained unchanged during the hydrolytic degradation assay, is that while in solution (prior to and during membrane formation) bis(2-ethylhexyl) phosphate acts as a catalyst for polymer hydrolysis. This unexpected result provides yet another advantage for this method: the possibility of tailoring the polymer degradation rate through the manipulation of the nucleating agent concentration and/or through its combination with another nucleating agent (such as stearic acid).

4. CONCLUSIONS Alternating soaking was successfully applied in asymmetrically porous poly(ε-caprolactone) membranes. Amphiphilic atoxic molecules, such as stearic acid and bis(2-ethylhexyl) phosphate, have proven to be effective nucleating agents for calcium

Figure 5. MTT assay results, in terms of PCL-A and PCL-E membrane absorbance at a 570 nm wavelength, after the alternating soaking cycles. Neat PCL was used as a reference. Samples marked with * are statistically different from the PCL reference based on the Bonferroni test at a 95% confidence interval. G


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phosphate growth because they enhanced calcium phosphate deposition on the membrane surface without any kind of surface pretreatment. Scanning electron microscopy showed that the nucleating agent played an important role in determining the calcium phosphate nucleation, growth, and morphology. On the one hand, stearic acid promoted complete surface coverage with the formation of spherical particles in only 10 soaking cycles. On the other hand, the use of bis(2ethylhexyl) phosphate resulted in the sparce deposition of CaP platelet particles, which could suggest that it is not as effective as stearic acid. However, further investigation must be performed concerning the optimal conditions for the adequate application of this nucleating agent. In conclusion, these nucleating agents have proven to effectively anchor and nucleate CaP onto polymeric surfaces through the production of permanent and available negative surface charges. Therefore, this new, simple, and atoxic method could be extended to a wide range of polymers and could be used in different applications.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.langmuir.6b04562. Description of the experimental design to optimize the alternating soaking method. SEM micrographs of the PCL top surface, bottom surface, and cross section. SEM micrographs of PCL-A top and bottom surfaces as well as the cross section after the alternating soaking cycles. Additional tables and figures. (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Laura C. E. da Silva: 0000-0002-9191-3440 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by Coordenaçaõ de Aperfeiçoá mento Pessoal de Nivel Superior (CAPES), Fundaçaõ de Amparo à Pesquisa do Estado de São Paulo (FAPESP), and ́ Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq) through Inomat, National Institute (INCT) for Complex Functional Materials.

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REFERENCES

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Amphiphilic Nucleating Agents to Enhance Calcium Phosphate

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