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Fabrication of novel open-cell foams of poly (#-caprolactone) / poly (lactic acid) blends for tissue engineering scaffolds Zirui Lv, Na Zhao, Zeming Wu, Changwei Zhu, and Qian Li Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b02233 • Publication Date (Web): 06 Sep 2018 Downloaded from http://pubs.acs.org on September 7, 2018

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Industrial & Engineering Chemistry Research

Fabrication of novel open-cell foams of poly (Ɛ-caprolactone) / poly (lactic acid) blends for tissue engineering scaffolds Zirui Lv1,3, Na Zhao2,3*, Zeming Wu2,3, Changwei Zhu4, Qian Li2,3 1 2

School of Materials Science & Engineering, Zhengzhou University, Zhengzhou 450001, China

School of Mechanics & Engineering Science, Zhengzhou University, Zhengzhou 450001, China

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National Center for International Joint Research of Micro-Nano Molding Technology, Zhengzhou University, Zhengzhou 450001, China 4

Sirade Technologies Inc., Toronto L4E 0S7, Canada *Corresponding Author: [email protected]

Abstract: Poly(Ɛ-caprolactone) / poly (lactic acid) (PCL/PLA) blends are very promising materials with biodegradable characteristics and tailorable performance for many applications. In this study, PCL and PLA were compounded at various ratios using a co-rotating twin-screw extruder. The morphology showed that they were immiscible but were dispersed well in each other. Very interesting and peculiar open-cell structures were obtained through a batch foaming process. Interconnected holes with flexible PCL fibrils were created by high tensile stress during cell expansion, which contributed to the rapid diffusion of CO2. No cells collapsed at high foam expansion under all foaming conditions. Moreover, a small diameter tubular PCL/PLA foamed scaffold had a tensile toe region of approximately 40 %, which indicated a potential application for vascular tissue engineering. The human umbilical vein endothelial cells cultured on the surfaces of PCL/PLA blend foams showed high viability and migration. Keywords: poly (Ɛ-caprolactone), poly (lactic acid), foams, scaffold, mechanical properties, cell culture

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Industrial & Engineering Chemistry Research 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

INTRODUCTION During the last decades, many biomaterials and different approaches have been used to fabricate the tissue engineering scaffolds1. Scaffolds are required to temporarily replace the morphology and mechanical function of a living tissue to be able to regenerate the tissue2. Currently, the main challenge is to fabricate three dimensional (3D) highly porous and interconnected scaffolds with sufficient mechanical properties that promote the adhesion and migration of human cells3. Interconnected pores are beneficial for cell infiltration and transportation of nutrients and waste. However, the mechanical properties strongly correlate with a 3D highly connected open structure that can provide support for the host tissue during cell growth on the scaffold. Many processing technologies have been used to fabricate the biomaterials into open cell structures, including gas foaming4-6, thermal induced phase separation7-10, particle leaching and solvent casting2, 11, and 3D bioprinting12. Among them, gas foaming with carbon dioxide (CO2) is an environmental friendly method to produce 3D porous materials without using an organic solvent. Normally, closed cells were obtained. To create a open cell structure, blending two immiscible polymers with hard and soft components by gas foaming can promote cell opening and can prevent cells from collapsing 13, 14. Peng Yu et al. fabricated bimodal open cell foams of poly (lactic acid) (PLA) / poly (butylene succinate) (PBS) blends via a two-step depressurizing batch foaming process using a hard/soft inhomogeneous system concept15. An Huang et al. fabricated a highly interconnected structure of PCL scaffold by leaching off 50% PEO by injection foaming PCL/PEO (50/50) blend3. Long Wang et al. produced the 18-fold expansion of isotactic polypropylene (iPP) / cellulose nanofiber (CNF) nanocomposite foams via a core-back injection molding technology. High alignment of CNFs reinforced the local melt strength and formed hybrid shish-kebab structures to prevent cells from collapsing. Thus, highly open-cell structures of iPP/CNF foams were produced16. In this study, we investigated the fabrication highly interconnected porous PLA/PCL blend scaffolds through a one-step batch foaming process using a hard/soft nonhomogeneous system. The cell opening mechanism was also investigated. PLA is one of the most promising biomaterials, but poor toughness and brittleness limit its applications. Blending with soft polymers can improve the toughness of PLA17-19. PCL have a low elastic modulus and a high elongation at break can be a good choice 20. In previous studies, polymer composites showed good performance for many applications21-32. Therefore, PCL/PLA blend with strong synergistic improvement in mechanical properties have an potential application for tissue engineering scaffolds. 2


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In this work, we proposed PCL as the matrix and PLA as the minor phase to fabricate blend (70/30) foams with highly interconnected pores by batch foaming process. PCL/PLA blends were prepared by twin screw extruder. Morphology of blends and its foams was investigated by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Tensile tests of the PCL/PLA foam scaffolds were also performed. Futhermore, the biological tests were carried out in culture of human umbilical vein endothelial cells (HUVECs) seeded on the surface of foam scaffolds.

EXPERIMENTAL SECTION Materials PCL (CAPA6500) with a molecular weight of 50,000 g/mole was purchased from Perstorp Polyols Inc. The glass transition temperature and melting temperature were 60 and 57 ℃, respectively. Its specific gravity was 1.13 g·cm-3. PLA (4032D) was purchased from NatureWorks LLC. Its specific gravity was 1.24 g·cm-3 and its molecular weight was 200,000 g/mole. The glass transition temperature and melting temperature were 62 and 169 ℃, respectively.

Sample Preparation PCL/PLA blends with mass ratios of 20/80, 30/70, 40/60, 50/50, 60/40, 70/30, 80/20, and 90/10 were compounded by a co-rotating twin screw extruder (screw diameter 27 mm and L/D ratio 42). The barrel temperatures were controlled in eight zones and a strand die with temperature profile ranging from 80 to 170 ℃. The extrude strands were cooled in a water bath and pelletized for the subsequent foaming process. The PCL/PLA (70/30) blend was pressed at 190 ℃ into a film (thickness is 0.3 mm). The films were cut into 20 ×15 mm pieces, which were folded into a tubular form with a diameter of 3.5 mm and were covered with aluminum foil. Then, a one step batch foaming process was carried out. The foaming experiments were measured five times for each set.

Characterization To analyze the morphologies, frozen unfoamed and foamed samples were prepared by a razor blade. Then, the cross sections were coated with a thin layer of platinum via a sputter coater. The morphologies were investigated by a scanning electron microscope (Keysight 8500, USA) and an atomic force microscopy (AFM) (Keysight 7500, USA). SEM images of PCL/PLA foam morphologies were analyzed using ImageJ software to estimate the particle diameters and standard deviation. The smooth surfaces of the 3



AFM samples were prepared using a ultramicrotome at room temperature, and the observations were conducted in a tapping mode at room temperature. The tensile test was carried out in accordance with the standard of cardiovascular implants and vascular prosthesis (ISO 7198). Speciemens of the PCL/PLA (70/30) blend foams (length 10 ± 1 mm and wall thickness 0.6 ± 0.2 mm) were tested at room temperature by a universal testing instrument (SUNS LTD CHINA) with a constant speed (2 mm/min). Eight tensile specimens were tested for each set. Human umbilical vein endothelial cells (HUVECs) were purchased from Oligobio (Beijing) and cultured in Roswell Park Memorial Institute (RPMI 1640) with the addition of 10 % fetal bovine serum (FBS). Scaffolds were soaked in 75 % medicinal alcohol for 12 hours, and then washed with PBS three times. Afterwards, they were soaked in PBS for 12 hours, and sterilizing UV light for 2 hour before cell seeding. Then, placed them on the bottom of 48-well tissue-cultured polystyrene (TCPS) plates. After that, HUVECs were counted and seeded at the density of 4,500 cells/cm2 on each sample. The culture plates were then placed in a incubator with 5 % CO2 at 37 ℃. The Live/Dead Viability/Cytotoxicity Kit were purchased from Invitrogen. Calcein AM (showing green) and propidium iodide (showing red) were used to stain the living and dead cells to quantify the biocompatibility and toxicity of PCL/PLA foamed scaffolds, respectively. Cell morphologies were determined by CF™ 568 phalloidin (red F-actin) staining. HUVECs were pretreated with 3.75 % formaldehyde solution and 0.1 % Triton-X, and then blocked by bovine serum albumin (BSA). The cells were incubated in 5 µl fluorescent phalloidin stock solution with 200 µl PBS for 20 minutes and 200 µl 4’, 6-diamidino-2-phenylindole (DAPI) for 10 minutes. After staining, the labelled HUVECs were observed under a fluorescent microscope (LEICA DMI3000 M, Germany). The CCK8 assay (Japanese colleagues) was used to caculate the numbers of cells living. 20 µL CCK8 dissolved in 200 µL RPMI 1640 was added to six samples for each test. After incubating for 2 hours in the incubator, 100 µL of the medium containing CCK8 was added to a 96-well plate. The numbers of cells living were then caculated via measuring the absorbance at 450 nm. Cell viability, proliferation and morphology were investigated at 1, 3, and 5 days.

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RESULTS AND DISCUSSION Immiscibility of PCL and PLA

Figure 1 SEM images of the cross-sections of the PCL/PLA blends etched by glacial acetic acid (a to d) and sodium hydroxide solution (e to h); Atomic force microscopy images of the PCL/PLA blends (i to l)

Figures 1a to d show the morphologies of the PCL/PLA blends (PCL content ≦50 %) etched off the PCL phase with 5 % glacial acetic acid solution for 8 hours at 37 ℃ 33, 34, and figures 1e to h show those of the PCL/PLA blends etched off the PLA phase with 5 % sodium hydroxide solution for 8 hours at 37 ℃35, 36. Figures 1i to l show the atomic force microscopy images of the PCL/PLA blends. Typical phase separation in all PCL/PLA proportions indicated that the PCL and PLA were immiscible. They were dispersed in the form of droplets in the corresponding PLA and PCL matrices, respectively37, 38. For instance, in the PCL/PLA (20/80) blend, the PCL particles of 0.25-2 µm in diameter were unformly dispersed in the PLA matrix (Figures 1a and 2a). With increasing PCL content, the average particle size increased from 0.5 to 1.10 µm and the particle size distribution was also broadened (Figure 2b and c). This was attributed to the competition between the two immiscible polymer particles, which broken up and coalesced during the thermal processing. Thus, co-continuous morphonologies of PCL and PLA (50/50 and 60/40) boends were formed (Figure 1d 5



to e). They were interpenetrating networks (IPN) with a few spherical particles when the droplets of the two immiscible polymers merged together. However, the co-continuous structures gradually disappeared and more particles with sizes ranging from 2.82 to 11.68 µm were observed (Figure 1f and l). Moreover, compared with the size of the PCL particles, the size of the PLA particles was larger (Figure 2d to f). This was related to a difference in viscosity ratio between those two blends which would promoted the foaming ability of PCL/PLA blends.

Figure 2 PCL pore size distribution in the PLA rich phase (a to c); PLA pore size distribution in the PCL rich phase (d to f)

Foam morphology of PCL/PLA blend PCL/PLA blend with weight ratio of 70/30 was chosen for fabricating open cell foams, and foaming parameters (saturation temperature, time and pressure) were investigated. All the prepared foams had peculiar open cell structures high porosity (Figures 3 to 5).

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Figure 3 SEM images of PCL/PLA (70/30) blend foams at various saturation temperatures (6.89 MPa, 1 hour)

Figure 3a to d show the morphologies of the PCL/PLA blend foams obtained at various saturation temperatures (36 to 42 ℃) at 6.89 MPa for 1 hour; Figures 3a’ to d’ show higher magnification SEM images. Instead of typical and disctinct open cell structures, cell walls are spider-web-like. Micrometer-sized holes with flexible PCL fibrils were randomly distributed on the cell walls, as indicated by the yellow circles. Also, some cell walls did not have fibrils (Figure 3c). This is because the PLA particles at low foaming temperature acted as defects that promoted cell opening. Therefore, the optimal foaming temperature was an very important fator for controlling the melt strength and therefore cell growth.

Figure 4 Schematic of the cell opening mechanism of PCL/PLA (70/30) blend

The cell opening mechanism of the PCL/PLA blend was subsequently investigated. During the foam expansion process, cell walls were subjected to tensile stress by the rapid diffusion of CO2. Therefore, holes with fibrils were formed during the cell growth. Once big holes were formed, the CO2 was rapidly released to the adjacent cells and the cells stopped growing immediately. The Mechanism of cell opening of PCL/PLA blend was shown in figure 4: 1) micrometer-sized holes and fibrils were 7



formed under the high tensile stress of the cell walls. 2) CO2 diffused off the cell walls, which further stretched them until the gas pressure was completely released. Normally, the stress on the cell walls was maximum in the center. A crack starts to form close to the wall center, then dissipates the stress by breaking holes4. In our work, the holes with fibrils were randomly dispersed on the cell walls (Figure 5). Because the PCL and PLA affect the cell growth in different ways, the cell wall start to rupture from thinner positions or the defects. PCL as a soft phase is torn into fibrils and PLA as a hard phase stops the crack growth. Until contacts with PCL/PLA interface, the process is terminated. Interestingly, no cell collapse at a high foam expansion. This indicted that the rupturing of the cell walls was rapid and occurred by a particular stretching ratio. Moreover, similar open cell structures were obtained at various saturation times, as shown in figure 6. Nanofibril structures mimicked the extracellular matrix (ECM), which would be suitable for cell attachment and proliferation.

Figure 5 SEM images of PCL/PLA (70/30) blend foams at various saturation times and temperatures (6.89 MPa)

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Figure 6 SEM images of PCL/PLA (70/30) blend foams at various saturation time (6.89 MPa, 42 ℃)

Mechanical properties of PCL/PLA foams

Figure 7 Stress-strain curves of the scaffolds: effects of the saturation temperature (a) saturation time (c); Young’s moduli of the scaffolds: effects of the saturation temperature (b) and saturation time (d)

To develop a small diameter vascular scaffold with adequate mechanical properties, tubular PCL/PLA foamed scaffolds (inner diameter 3.5 mm and wall thickness 0.6 mm) were fabricated. Effection of foaming temperature (Figure 7a; 13.79 MPa and 1.5 hour) and time (Figure 6c; 6.89 MPa and 38 ℃) on the tensile stress-strain curves are shown in figures 7a and c. Tensile properites of all the samples of showed a strain toe region of approximately 40% with a high modulus. The last region of the stress-strain curves is typical of polymeric foams and is attributed to the deformation of the cells. Compared with Young’s modulus of the human’s brain veins (1.72 MPa) and pig’s leg veins (7.69 MPa) 39, those of the PCL/PLA foamed scaffolds were suitable for vascular scaffolds (Figure 7b and d, 2.86 - 9.73 MPa) . Young’s modulus of the foamed scaffold prepared at 40 ℃, 13.79 MPa and 1.5 hour was 8.10 MPa, which approached that of pig’s leg veins. Moreover, Young’s modulus of the PCL/PLA foamed scaffolds did not change much with increasing saturation time, as 9



shown in figure 7d. Thus, the PCL/PLA foamed scaffolds meet the mechanical properties required for small diameter vascular tissue engineering applications.

3.4. Cell culture

Figure 8 Fluorescence staining (a to c), cytoskeleton (a’ to c’) and CCK8 (d) of HUVECs cultured on the scaffolds; Trance well picture of HUVECs through out of the PCL/PLA foamed scaffold (e)

To confirm the biocompatibility of the open cell PCL/PLA foamed scaffold, HUVECs were cultured on the surface of the scaffolds for days. Figures 8a to c show the living/dead results by confocal microscopy, indicating that the open cell structure of the scaffold had a high cell viability. All living HUVECs on the scffolds were were green. It indicated that PCL/PLA foam scaffolds had no cytotoxicity to HUVECs. It is known that if the shape of cells is flat and spread-out, they have strong interaction with the substrate. In contrast, if they are spherical, it is harder for them to be well attached to the substrate40-42. Figures 8a’ to c’ show that HUVECs on the surface of the scaffolds were well spread, confirming that an open cell structure with fibrils was favorable for cell attachment and proliferation43. According to CCK8 assay, more cells 10


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on the scaffold foams were observed (figure 8d), which was in accordance with the live/dead data. Figure 8e shows the trance well picture of the HUVECs after 2 days culturing on a 2-mm-thick substrate that was colored by crystal violet. Interestingly, the cells had a very clear pseudopod after two days, confirming the great potential of open cell structures with fibrils of the PCL/PLA foamed scaffold for applications in vascular tissue engineering.

CONCLUSION In this study, PCL/PLA foam blends with a peculiar open cell structure containing fibrils were fabricated via a one step batch foaming process. The morphology showed that the PCL and PLA were well dispersed in each other. The blend containing 70 wt. % PCL and 30 wt. % PLA had a high porosity after foaming, and the interconnected holes with flexible PCL fibrils were created owing to high tensile stress during cell expansion. No cells collapsed at a high foam expansion under all the foaming conditions. A small diameter PCL/PLA foamed tube had a tensile toe region of approximately 40%. Moreover, HUVECs cultured on the PCL/PLA foam scaffolds showed a high viability and migration. Therefore, the PCL/PLA foamed vascular scaffolds had a significant potential applications in vascular tissue engineering.

ACKKNOWLEDGEMENTS The authors would like to thank the China Postdoctoral Science Foundation (grant no. 2017M610460), the International Science & Technology Cooperation Program of China (grant no. 2015DFA30550), the China National Youth Science Foundation (grant no.11502238) and the Key Scientific Research Project of Henan Province (grant no.17A430030) for financial support of this work.

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Figure 1 SEM images of the cross-sections of the PCL/PLA composites etched by glacial acetic acid (a to d) and sodium hydroxide solution (e to h); the scale bar is 2 µm. Atomic force microscopy images of the PCL/PLA composites (i to l); the scale bar is 2 µm. 150x104mm (300 x 300 DPI)



Figure 2 a to c: PCL pore size distribution in the PLA rich phase; d to f: PLA pore size distribution in the PCL rich phase 152x86mm (300 x 300 DPI)


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Figure 3 SEM images of PCL/PLA (70/30) foams at various saturation temperatures (6.89 MPa, 1 hour). 150x74mm (300 x 300 DPI)



Figure 4 Schematic of the formation of PCL/PLA foams by batch foaming 570x193mm (300 x 300 DPI)


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Figure 5 SEM images of PCL/PLA (70/30) blend foams at various saturation times and temperatures (6.89 MPa) 150x100mm (300 x 300 DPI)



Figure 6 SEM images of the PCL/PLA (70/30) blend foams at various saturation time (6.89 MPa, 42 ℃) 150x50mm (300 x 300 DPI)


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Figure 7 Stress-strain curves of the scaffolds: effects of the saturation temperature (a) saturation time (c); Young’s moduli of the scaffolds: effects of the saturation temperature (b) and saturation time (d) 160x123mm (300 x 300 DPI)



Figure 8 Fluorescence staining (a to c), cytoskeleton (a’ to c’) and CCK8 (d) of HUVECs cultured on the scaffolds; Trance well picture of HUVECs through out of the PCL/PLA foamed scaffold (e) 495x467mm (150 x 150 DPI)


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