key: cord-0253337-5bm7kfwt
authors: Sun, Jie; Jing, Linzhi; Liu, Hang; Huang, Dejian
title: Generating Nanotopography on PCL Microfiber Surface for Better Cell-Scaffold Interactions
date: 2020-12-31
journal: Procedia Manufacturing
DOI: 10.1016/j.promfg.2020.05.090
sha: 03025d92bdc70cdb1c90e312fa87ff2d5eec2c4d
doc_id: 253337
cord_uid: 5bm7kfwt

Abstract To understand the behavior of cells in natural environment, 3D scaffolds are needed to provide biomimetic environment for routine cell growth in vitro. Electrohydrodynamic jet (EHDJ) printing, one of the emerging 3D printing techniques, is capable of fabricating 3D scaffolds with oriented fibers and customized microscale structures. However, most of the EHDJ-printed scaffolds lack of hierarchical structures such as nanotopographies present in native tissues. In this study, we propose a novel approach that combine EHDJ printing of poly(ε-caprolactone) (PCL)/gliadin composite inks to produce scaffolds with oriented microfibers. The nanoscale pores and flaws (fiber nanotopography) could be generated on the printed fibers by simply leaching the gliadin phase from the printed PCL/gliadin scaffolds. The density and size of nanotopographical features could be controlled by adjusting the gliadin ratio. Furthermore, by seeding mouse embryonic fibroblasts (NIH/3T3) cells, it clearly manifested that such scaffolds are favorable to cell adhesion, migration and proliferation efficiently. Thus, the newly designed PCL scaffolds with both micro- and nano-scale topographies can be a promising 3D cell culture platform with improved cell-scaffold interactions.

Since cells are capable of sensing mechanical rigidity in their surroundings, bioscaffolds' structure, mechanical and biochemical cues have been identified as the key factors to reestablish physiological cell-cell, and cell-extracellular matrix (ECM) interaction in vitro [1] . To create an artificial environment for cell culture, bioscaffolds should span features from micron to nanoscale for cell adhesion and proliferation with proper flow of gases, nutrients, and waste. Microstructural features on bioscaffolds can determine scaffold porosity and mechanical properties, and sufficient biomechanic performance may shield cells from damaging compressive or tensile forces. The microstructural features are also critical to ensure cell adhesion, as the size of many adherent somatic cell types typically spans a distance of 10-150 micrometer [2] .

Nanoscale features on scaffold surface determine cells' physical interaction and their response to a substrate (Bettinger, 2009) . For example, collagen fibrils are typically organised across a length scale of 50-200 nm and enable the adhesive interaction of fibroblast cells [3] . The rat calvarial osteoblasts grow significantly faster on the titanium coated epoxy substrate with increasing surface roughness, but human gingival fibroblasts show the opposite proliferation behavior [4] . Also, human mesenchymal stem cells spread and proliferate more actively on electrospun terephthalate composite fibers with a nanoporous surface than those with a smooth nanotopography [3] . Hence, desirable cell-culture scaffolds should possess not only appropriate microscale fiber features, but also nanoscale features like surface roughness

Since cells are capable of sensing mechanical rigidity in their surroundings, bioscaffolds' structure, mechanical and biochemical cues have been identified as the key factors to reestablish physiological cell-cell, and cell-extracellular matrix (ECM) interaction in vitro [1] . To create an artificial environment for cell culture, bioscaffolds should span features from micron to nanoscale for cell adhesion and proliferation with proper flow of gases, nutrients, and waste. Microstructural features on bioscaffolds can determine scaffold porosity and mechanical properties, and sufficient biomechanic performance may shield cells from damaging compressive or tensile forces. The microstructural features are also critical to ensure cell adhesion, as the size of many adherent somatic cell types typically spans a distance of 10-150 micrometer [2] .

Nanoscale features on scaffold surface determine cells' physical interaction and their response to a substrate (Bettinger, 2009) . For example, collagen fibrils are typically organised across a length scale of 50-200 nm and enable the adhesive interaction of fibroblast cells [3] . The rat calvarial osteoblasts grow significantly faster on the titanium coated epoxy substrate with increasing surface roughness, but human gingival fibroblasts show the opposite proliferation behavior [4] . Also, human mesenchymal stem cells spread and proliferate more actively on electrospun terephthalate composite fibers with a nanoporous surface than those with a smooth nanotopography [3] . Hence, desirable cell-culture scaffolds should possess not only appropriate microscale fiber features, but also nanoscale features like surface roughness

Since cells are capable of sensing mechanical rigidity in their surroundings, bioscaffolds' structure, mechanical and biochemical cues have been identified as the key factors to reestablish physiological cell-cell, and cell-extracellular matrix (ECM) interaction in vitro [1] . To create an artificial environment for cell culture, bioscaffolds should span features from micron to nanoscale for cell adhesion and proliferation with proper flow of gases, nutrients, and waste. Microstructural features on bioscaffolds can determine scaffold porosity and mechanical properties, and sufficient biomechanic performance may shield cells from damaging compressive or tensile forces. The microstructural features are also critical to ensure cell adhesion, as the size of many adherent somatic cell types typically spans a distance of 10-150 micrometer [2] .

Nanoscale features on scaffold surface determine cells' physical interaction and their response to a substrate (Bettinger, 2009) . For example, collagen fibrils are typically organised across a length scale of 50-200 nm and enable the adhesive interaction of fibroblast cells [3] . The rat calvarial osteoblasts grow significantly faster on the titanium coated epoxy substrate with increasing surface roughness, but human gingival fibroblasts show the opposite proliferation behavior [4] . Also, human mesenchymal stem cells spread and proliferate more actively on electrospun terephthalate composite fibers with a nanoporous surface than those with a smooth nanotopography [3] . Hence, desirable cell-culture scaffolds should possess not only appropriate microscale fiber features, but also nanoscale features like surface roughness and topography to ensure proper cell-scaffold interactions for cell adhesion, migration and proliferation.

Electrohydrodynamic jet (EHDJ) has recently drawn great attention for its capability to print micron scale fibers using biopolymer solutions. The printing resolution of EHDJ is improved by about two orders of magnitude in comparison to the conventional inkjet printing systems. The deposited fibers on the substrate can be orientated by a computer controlled precision stage and stacked into scaffolds with tailored microstructure. Most importantly, EHDJ enables the fabrication of biomimetic fibrous scaffolds with the feature size close to the scale of extracellular matrix, which can facilitate the cell attachment, proliferation and tissue regeneration [3] . This great potential motivates researchers to devote intensive efforts on the development of EHDJ technologies.

A few synthetic biopolymers such as poly-ε-caprolactone (PCL), polyethylene oxide (PEO), and polylactic acid (PLA), have been extensively utilized as bioink materials in EHDJ [5] . PCL is of particular interest by reason of its low melting point, superior solubility and printability. While, the pristine PCL surface lacks of sufficient signals for cell recognition and adhesion [6] .

As the surface nanotopography is proven to have significant influence on cellular attachment, motility, proliferation, and infiltration [7] , a possible solution is to modify the PCL scaffolds' fiber surface to enhance cellscaffold biointerface. Usually toxic chemicals or synthetic nanoparticles are applied for surface modification, while it is not desirable for downstream biomedical applications. An alternative is to blend PCL with biopolymers such as collagen [8] and silk fibroin [9] to improve the cytoaffinity of the biointerface. However, the batch-stability and potential immunogenicity are of concern when using animal-derived products.

Plant proteins become prevailing because of their definitive molecular integrity, abundance, and free of transmitting zoonotic diseases [10] . Recently, our group has developed PCL/zein composite inks for EHDJ printing, and the zein containing scaffolds manifest an improved cytoaffinity in cell culture studies [11] .

We expand our search to gliadin, one of the major gluten storage proteins of wheat. It can self-assemble into nanoparticles in certain solvents but dissolve in water [12] . In this study, gliadin is blended with PCL to prepare PCL/ gliadin composite inks for scaffold fabrication via EHDJ technique. The nanotopography surface is generated on printed microscale fibers by simply solution leaching. The morphology and tensile properties of produced scaffolds are characterized, and cellular behaviors are investigated by seeding mice embryonic fibroblast cells.

Even though the EHDJ mechanism is conceptually simple, the actual formation process strongly depends on the properties of the inks (viscosity, surface tension, and electrical conductivity), environmental factors (temperature and humidity), and other process parameters (nozzle to substrate distance, solution feeding rate, and dimensions of nozzle). To achieve stable EHDJ fabrication, a real-time monitoring system is needed to observe jet triggering and detect any abnormal modes. This would facilitate to optimize process parameters for different composite inks and ink properties, and support the control of morphologies of deposited fibers and scaffolds. Meanwhile, special PCL/gliadin composite inks would be developed for EHDJ fiber producing, followed by a gliadin removing process to generate nanotopography patterns on fiber surface eventually.

EHDJ can fabricate fibrous scaffolds with micron scale fibers in a controllable and reproducible manner with low cost and high efficiency. Fig. 1 shows a schematic diagram of a solution-based EHDJ printer and the jet printing process. As shown in Fig. 1 , our EHDJ setup consists of a solution feeding system, a high voltage power supply (output DC voltage from 0 to 10 kV), and a 3-axis precision motorized stage. The solution feeding system consists of a syringe pump (New Era Pump System), a syringe (internal diameter = 13 mm, volume = 5 ml), a flexible hose, and a nozzle with internal diameter of 0.5 mm. Gliadin and PCL pellets with an average molecular weight of 50 kDa are sequentially dissolved in the glacial acetic acid (> 99.7% pure) to generate a series of homogenous composite inks. In total, PCL ink solution (70 w/V%, g/mL), and two types of PCL/gliadin ink solutions including PCL/gliadin-10 (60 w/V% PCL, 10 w/V% gliadin, g/mL), and PCL/gliadin-20 (50 w/V% PCL, 20 w/V% gliadin, g/mL) are prepared for scaffold fabrication studies.

The high voltage power supply is applied to generate applied voltage (V) between the nozzle and the substrate. This voltage should be above a certain threshold to trigger and maintain EHDJ process. Higher voltage may lead to smaller fiber diameter, however, beyond an optimum voltage, the fiber diameter may start to increase due to reduction in flight time [5] . It is set around 3KV in this study. The precision stage, from Aerotech Company (Pittsburgh, PA USA), is driven by linear motors. It has a travel distance of 150 mm with 3 μm accuracy on X and Y axes, and 50 mm with 5 μm accuracy on Z axis. The substrate, a polished silicon wafer, is fixed on X-Y plane. The stage speed along X and Y axes are set the same ranging between 100mm/s and 300mm/s. The moving stage along X and Y axes could generate a mechanical drawing force to guide the deposition of the EHDJ fiber, which significantly affected the fiber size, pattern and position.

The mechanical drawing force acting on the jetting increases with the stage speed. This leads to an increasing degree of slant jet and its elongation rate, and eventually generates diverse trajectory. When the stage speed is greater than the downward speed of the jet, the fiber length deposited on the substrate is larger than the jet length fallen on substrate per unit time [5] . Moreover, the fiber orientation can be controlled by changing the above mentioned manufacturing parameters.

As the printing resolution moves into micro to nano scale, a slight fluctuation of environmental factors such as temperature, humidity, air flow and stage vibration might affect the printing accuracy. Also, it takes time to achieve and maintain a steady flow rate especially for high viscous biopolymer solutions. In order to ensure the printing quality, it is very necessary to establish the relationship between the cone shapes and the stability of scaffold fabrication process.

Since the EHDJ jet velocity would significantly lower down for high viscous composite biopolymer solutions, we use digital microscopic imaging technique, where the EHDJ process is video recorded by a Supereyes B011 digital microscope with 200 magnifications and 30 frames per second to monitor the real time jet formation and deposition processes. The camera position and shooting angle are calibrated by aligning the area of the grayscale nozzle image with a predefined nozzle position [13] . Fig. 2(a) shows a captured standard EHDJ cone shape with a straight jet. Fig. 2(b) shows a cone jet with helical deformation which consists of two distinct parts: a long, roughly vertical "tail" (with length in millimeters, and diameter in micrometers to nanometers) which deforms primarily by severe electrostatic force stretching, and a helical "coil" in which the deformation is dominated by bending and twisting. Fig. 2(c) shows the discharge phenomenon for the PCL/gliadin composite inks in EHDJ. This could be attributed to the charged amino acid residues in the peptide chains of gliadin, which alters the electrical properties of the supplied composite inks. 

The scaffold is constructed by stacking the printing fiber layer directly on the top of each other, and eventually forming a 3D scaffold structure. The printing time is mainly dependent on the size of scaffold and the stage speed. It usually takes around 30 min to print a 4 × 4 cm scaffold with 200μm pore size and 12 layers by running the stage at an average speed of 150 mm/s. Surface morphology of the fabricated PCL scaffolds is imaged using a scanning electron microscope (SEM, JSM-6510, JEOL, Japan). Fiber width, height, pore size and the overall thickness are measured, and the final value is an average of ten measuring points. A discharge phenomenon is observed for gliadin containing inks at initial jetting process as Fig. 2(c) , which is quite similar to the phenomenon observed when using PCL/zein composite inks. While this does not happen to PCL ink. The charge could be released properly when the stage runs at a rational speed in PCL/gliadin scaffold fabrication through the process parameters' optimization.

Taking advantage of the good solubility of gliadin in aqueous and ethanol [12] , nanoscale pores and flaws could be created on fiber surface by washing with deionized (DI) water and dried in vacuo. The dynamic weight loss of PCL/gliadin-10 and PCL/gliadin-20 scaffolds was plotted in Fig 3. It can be seen that PCL/gliadin scaffolds lose their weight significantly within 20 hours, and the weight loss of PCL scaffold is negligible. The weight loss curves of PCL/gliadin-10 and PCL/gliadin-20 tend to converge after 20 hours, and it means that most of gliadin is washed off from fiber surface.

The weight loss of PCL/gliadin scaffolds is mainly attributed to the releasing of protein aggregates. The amount of the weight loss is almost consistent for a specific ratio of the gliadin portion in the composite. Thus, surface engineered nanoporous scaffolds, namely PCL-10-D and PCL-20-D, are produced from fabricated PCL/gliadin-10 and PCL/gliadin-20 scaffolds respectively. There is no obvious deformation on the PCL-10-D and PCL-20-D scaffolds during this post processing.

Such PCL-D scaffolds are used to study the influence of surface nanotopography on cellular behaviours in a 3D culture mode. The corresponding morphological changes of the fiber surface before and after treatment are illustrated in Fig. 4 . Numerous nanosized pores and flaws are generated on the fiber surface of PCL-D scaffolds. The average sizes of nanopores (indicated by red arrows in Fig. 4 ) generated on the fiber surface of PCL-10-D and PCL-20-D scaffolds are 89.2 ± 50.7 nm and 109.1 ± 66.9 nm respectively, and some of pores interconnect with each other to form larger longitudinal flaws indicated by dashed boxes, ranging from few hundred nanometers to a few microns in length. This is mainly attributed to the dissolution of protein aggregates, resulting the nanoscale pores and flaws on the fiber surface. Nanopores may be generated inside fibers when are channelled to the fiber surface, which can provide more space for cell adhesion. The density of nanopores and flaws on PCL-20-D scaffold is clearly higher than that on PCL-10-D one, which suggests the nanotopographical features of fibers could be manipulated by controlling the ratio of the gliadin in the composite inks. 

Fibrous scaffolds are architecturally characterized by features such as fiber diameter, pore size and internal configuration of deposited fibers. These features play an important role in determining the overall scaffold mechanical properties, in additional to the inherent material strength. The microscale morphology of the scaffolds is characterized by SEM and the representative images of PCL/gliadin-20 and PCL-D-20 scaffolds are illustrated in Fig. 5 (a) and (b) . The scaffold model is shown in Fig. 5(c) . The geometries in Fig. 5  (a) and (b) are not exactly the same due to taken from slightly different viewpoints.

All scaffolds have well defined lattice-work microstructures with an average pore size around 177 μm. The side walls are composed of 12 layered fibers and the average diameter of printed fibers is approximate to 10 μm. As shown in Fig. 5(b) , the integrity of gliadin containing scaffolds still remains even after the gliadin is leached. We further compare the porosity of the scaffolds since it plays a vital role on cell-scaffold interactions and nutrient-waste exchange. The porosities slightly rise up to 90.3 ± 0.3 % and 91.3 ± 0.9 % for PCL-10-D and PCL-20-D scaffolds respectively, compared with 89% for PCL scaffold. 

The tensile properties of the scaffolds are examined by using a universal testing machine (HD-B609B-S, HAIDA, China). The testing is performed by stretching with an initial gauge length of 20.0 mm at a speed of 1 mm/min and 10 mm/min for pre-loading and loading conditions respectively. The stress-strain curve of PCL, and PCL-D scaffolds is illustrated in Fig. 6 . In general, the PCL scaffold shows a typical amorphous polymer behaviour with a prolonged strain hardening phase and large breaking strain, which suggests that PCL is a ductile material with superior toughness.

The tensile behaviour of PCL-10-D scaffold overall resembles to that of pristine PCL one except for minor increase of yield stress and strain. While, the ultimate stress and strain of PCL-20-D scaffold dramatically drop to 8.4 MPa and 304.2 % respectively, which are much smaller than that of the PCL one. The Young's modulus of PCL-20-D scaffold also decreases by around 30 %. PCL-10-D scaffold shows minor changes in tensile properties compared with PCL ones. The fiber surface of PCL-10-D scaffold is mainly composed of isolated nanopores with average size around 90 nm, which could be compressed during stretching and put an insignificant impact on the bulk property of PCL phase. While, PCL-20-D scaffolds is apparently brittle. A growing number of larger flaws with size up to a few microns on the fiber surface of PCL-20-D scaffold are observed, which makes it easy to be ruptured in tensile test. This is consistent with the report that the gliadin could self-assemble into either nanosized spheres, rods, or prolate ellipsoids because of the amphiphilic nature of polypeptide chains in solution [12] . Thus, the proposed method is capable of controlling scaffold's mechanical behaviors by adjusting the gliadin ratio, and PCL-10-D is supposed to be a competent alternative of pristine PCL scaffold with the improved surface nanotopography and similar tensile properties.

The PCL-10-D and PCL-20-D scaffolds, are applied to study the influence of surface nanotopography on cellular behaviours in 3D culture mode. The morphologies of cell seeded scaffolds are visualized by confocal laser scanning microscopy (CLSM, LSM-880, ZEISS, Germany). Statistical analysis of the data was done using student's t test by GraphPad Prism software (version 5.01).

To investigate the influence of the fiber nanotopography on cellular behaviours in a 3D culture mode, the PCL-D scaffolds and pure PCL scaffolds are applied to grow NIH/3T3 cells. For initial cells seeding, a small volume of NIH/3T3 cells suspension (30 μL, density 1.7 × 106 /mL) is pipetted on each scaffold in a 24 well ultralow attachment culture plate to allow the cells to contact with the surface of scaffolds. After incubation for 3 hours, the remaining culture medium (480 μL) is added in each well. Some cells start to partially adhere on the fibers of scaffold whereas the unattached cells gathered together to form the clusters, dispersing in the medium. The morphologies of cells seeded scaffolds are visualized by CLSM and the amounts of cells on scaffolds at the 1st, 4th and 7th day of culture are evaluated by MTS method (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, USA) as shown in Fig. 7 .

The cell counting results at the 1st day show that the numbers of cells attached on PCL-D scaffolds are approximate three times higher than that on the PCL scaffold. This indicates that the porous fibre surface with nano-pores and flaws are in favour of cell attachment. Subsequently, the cells start to spread, migrate, and proliferate on the scaffolds, and the cell numbers on PCL -D scaffolds are about two times higher than that on PCL at the 4th day of culture. This is consistent with reported studies that cells preferentially grow on moderately rough substrates [9] .

As shown in Fig.7(b) , cells close to the intersection of orthogonal fibres are found to be capable of elongating as spindle shape and accommodating themselves in the cavity with extended filopodia at leading edge grabbing on the adjacent fibres. As a consequence, the fibroblasts actively proliferate from periphery side walls to the centre of the cavity, forming a unique circular structure as Fig. 7(a) . There is no significant difference in cell numbers on PCL-10-D and PCL-20-D scaffolds at the 1st and 4th day of culture, which suggests the engineered nanotopography on PCL-10-D might be sufficient to boost the cell adhesion and growth. Although the cells adhesion and growth on scaffolds initially is not homogeneous because of the uneven cell seeding density, at the 7th day of culture, cells could migrate across the adjacent pores, take over all cavities eventually as shown in Fig. 7(d) . The number of cells on scaffolds reach saturated, which are approximate twenty times higher than that of the initial seeding. The cells go into a stationary growth state and form a tissue-like cell sheet thoroughly. The cell-seeded PCL, PCL-10-D and PCL-20-D scaffolds on day 1 is shown in Fig. 8 . The cells tightly touch to the engineered fiber surface of PCL-D scaffolds with extensive filopodia in contact with the nanopores and flaws highlighted in Fig. 8 (a) and Fig. 8 (b) with red dashed box. In contrast, the leading edge of fibroblasts adhered on the surface of PCL seems to be more clear and smooth, indicating a loose cellsubstrate interaction as shown in Fig. 8 (c) . In other words, cells could detect the topographical features of surrounding environment by highly dynamic filopodia [14] . Subsequently, the cells would spread on the fiber surface, migrate spatially between the fibers. Also, the attached cells might in turn affect the nanotopography of the contacting surface as it becomes much rougher than the primitive surface. 

In order to produce features found naturally within ECM and enhance cell differentiation, scaffold based cell culture is growing dramatically. Fibrous scaffold structures are applied in 3D cell culture, which create an artificial environment so that cells can interact with their surroundings in all three dimensions.

In this study, the PCL/gliadin composite inks have been used in EHDJ-printed scaffold fabrication with oriented microfibers and gliadin serves as a template to generate nanostructured pores by leaching treatment. The density and size of these pores and flaws could be tuned by controlling the gliadin ratio in the composite. The tensile test shows the PCL-D scaffolds resemble to that of pristine PCL scaffold except for dramatic decline of ultimate strain of PCL-20-D scaffold.

The SEM images of fiber surface morphology indicate that the gliadin is the sacrificial template to generate fiber nanotopography and it probably existes as nanosized aggregates in the PCL/gliadin inks. The cell adhesion and proliferation rate, tested on NIH/3T3 cell lines, indicate that the fiber nanotopography alters cellular behaviors and enhance cell-scaffold interactions. Follow-up studies will investigate how the fiber nanotopography influences the cellular functions and apply the established 3D cell culture models for cytotoxicity test or anti-cancer drug screening. Meanwhile, gliadin may mix with other biocompatible polymers to fabricate scaffolds with nanostructured surface for certain applications. Meanwhile, we also plan to add a second syringe pump for cell or drug seeding along with the scaffold fabrication process for more advanced applications in biology studies and drug screening.

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