Biomedical Microdevices

, Volume 12, Issue 5, pp 841–848

The fabrication of PLGA microvessel scaffolds with nano-patterned inner walls

Authors

    • Department of Mechanical EngineeringNational Chung-Hsing University
    • Institute of Biomedical EngineeringNational Chung-Hsing University
  • Yan-Cheng Lin
    • Taiwan Semiconductor Manufacturing Company
  • Shan-hui Hsu
    • Institute of Biomedical EngineeringNational Chung-Hsing University
    • Institute of Polymer Science and EngineeringNational Taiwan University
Article

DOI: 10.1007/s10544-010-9438-x

Cite this article as:
Wang, G., Lin, Y. & Hsu, S. Biomed Microdevices (2010) 12: 841. doi:10.1007/s10544-010-9438-x

Abstract

Poly (lactic-co-glycolic acid) (PLGA) is one of the most commonly used biodegradable, biocompatible materials. Nanostructured PLGA has immense potential for application in tissue engineering. In this article we discuss a novel approach for the fabrication of PLGA microvessel scaffolds with nanostructured inner walls. In this novel nano-patterning approach, the thermal reflow technique is first adapted to fabricate a semi-cylindrical photoresist master mold. A thin film of titanium and a thin film of aluminum are sputtered in sequence on the semi-cylindrical microvessel network. Aluminum foil anodization is then executed to transform the aluminum thin film into a porous anodic aluminum oxide (AAO) film. During the casting process a PLGA solution is cast on the AAO film to build up semi-cylindrical PLGA microstructures with nanostructured inner walls after which inductive coupled plasma (ICP) is implemented to assist bonding of the two PLGA structures. The result is the building of a network of microchannels with nano-patterned inner walls. Bovine endothelial cells (BECs) are carefully cultured in the scaffold via semi-dynamic seeding for 7 days. Observations show that the BECs grew more separately in a nano-patterned microvessel scaffold than they did in a smooth surface scaffold.

Keywords

Microvessel scaffoldPLGANanostructureCircular microchannelsBovine endothelial cells

1 Introduction

During the past decade, the cross-disciplinary integration of engineered extracellular matrices (typically called scaffolds), cells, and biologically active molecules has led to the fabrication of artificial tissues in the laboratory (Langer and Vacanti 1993). In this process cells are usually implanted or cultured in a biodegradable scaffold capable of supporting three-dimensional tissue formation. Such scaffolds serve a number of purposes, as the foundation for cell attachment and migration, for the exchange of nutrients, and delivering and retaining of the cells and biochemical factors. One of the continuing, persistent challenges confronting tissue engineering is the lack of intrinsic microvessels for the transportation of nutrients and metabolites, thus making it difficult for any implanted cell to acquire sufficient oxygen and nutrients to function properly. One feasible solution to this problem is to provide an artificial microvascular system capable of enabling the regular operations of metabolism.

Borenstein’ group combined the techniques of semiconductor fabrication and the deep reactive ion etching (DRIE) to make microvessel scaffolds on PolyDiMethylSiloxane (PDMS). Endothelial cells of human microvessels (HMEC-1) were successfully seeded in the scaffolds (Fidkowski et al. 2005; Borenstein et al. 2002; Shin et al. 2004). Moldovan’s team (Moldovan and Ferrari 2002; Kulkarni et al. 2004) looked at new microvascular structures which would enable in vivo transfer by implantation. Wang et al. (2005) used stainless steel electroforming and silicon electroforming approaches to fabricate microvascular scaffolds on polycarbonate (PC) and poly lactide-co-glycolides (PLGA) for the cultivation of bovine endothelial cells (BEC). Computer simulations designed to optimize the structure of microvascular scaffolds for better cell-adhesion have also been conducted (Wang et al. 2006; Wang and Hsu 2006). In efforts to solve the dead volume problems usually encountered in rectangular microchannels, the same group has fabricated microvessel scaffolds with circular microchannels on both PDMS and PLGA substrates (Wang et al. 2007a, b).

Since the microvessel scaffolds are eventually meant to be implanted in a living body, biodegradability and biocompatibility are the main considerations. There have been many biomaterials investigated for that purpose and many are now commercially available for tissue engineering scaffolds. Among them, PLGA, consisting of polylactic acid and polyglycolic acid, is one of the most successful, because of its good biodegradability and biocompatibility. Nano-patterned PLGA has even greater potential in tissue engineering. Many promising nano-patterned PLGA biomaterials have been developed for tissue engineering. For example, Park et al. (2005) demonstrated that NaOH-treated PLGA scaffolds enhanced chondrocyte functions more than did non-treated scaffolds. Webster et al. (Webster et al. 2005; Savaiano and Webster 2004) compared the adhesion of pseudomonas fluorescens on nanophase with that on conventional grain size alumina substrates. Miller et al. (2006) reported that the patterning of the PLGA with nanometer spherical features promoted cell spreading and adhesion. Min et al. (2004) discovered that PLGA and PLGA/chitin matrices are good for normal human fibroblasts. However, the applications of the existing nano-patterned methods for PLGA materials are limited to the open-space scaffolds. The task of fabricating enclosed scaffolds (such as microvessel scaffolds), and of patterning nanostructures on the inner walls of microchannels (with circular cross-sections) is still challenging.

In this study, we focus on the fabrication of PLGA microvessel scaffolds with nanostructured inner walls for which we develop a novel nano-patterning approach. The thermal reflow technique was first adapted to fabricate the semi-cylindrical photoresist master mold. An aluminum thin film was then sputtered on this master. Anodic anodization was conducted to transform the aluminum thin film into a porous AAO film. A PLGA solution was prepared by dissolving PLGA polymer in acetone. This was then used in a casting process on the AAO film to produce a semi-cylindrical PLGA microstructures with nanostructured inner walls. Finally the two PLGA membranes were bonded together to form microstructures consisting of circular microchannels that have nanostructured inner walls.

2 Materials and methods

2.1 Materials

The procedure for fabrication of the nano-patterned microvessel scaffolds includes the implementation of a thermal reflow technique to build the semi-cylindrical photoresist mold, sputtering of the aluminum thin film and preparation of the PLGA anodization solution, casting of the PLGA solution onto the semi-cylindrical photoresist mold, stripping off the PLGA membrane from the photoresist mold, and utilizing ICP to assist in the bonding of the two PLGA structures together to form a network of microchannels with nano-patterned inner walls.

2.1.1 Nano-porous semi-cylindrical photoresist replica mold fabrication

The manufacturing procedures used to produce the nano-patterned semi-cylindrical photoresist master mold are schematically illustrated in Fig. 1.
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Fig. 1

Manufacturing procedures for the nano-patterned semi-cylindrical photoresist replica mold

  1. (1)
    Exposure: The aluminum foil (99.9995%, 175 μm thick) was cleansed using ethanol, acetone, and deionized water (DI water) in sequence. An electropolishing process was then carried out with an electrolyte solution of perchloric acid (60 wt %) and ethanol (99.5 wt %) (the mixing ratio was 1:5) under a constant voltage of 20 V for 2 min to further polish the surfaces of the foil. A 60 μm thick layer of JSR THB-120 N negative photoresist was spin-coated onto the polished aluminum foil (at 350 rpm for 10 sec followed by 500 rpm for 25 sec). The reason that the THB-120 N negative photoresist spin-coating process was implemented was to produce the relatively thick film needed to satisfy the high aspect ratio requirement of microvessel scaffolds. This made it easier to form a semi-cylindrical microstructure. The photoresist was then exposed to UV light for 10 min using the photomask as shown in Fig. 2.
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    Fig. 2

    Photomasks for the microvessel scaffolds

     
  2. (2)

    Development: The development process (using the developer solution THB-D1) was then conducted to transfer the microvessel network patterns onto the photoresist. Since the size of the bovine endothelial cells (BEC) for cell seeding is about 10–15 μm, the diameter of each microchannel ranged from 60 μm to 120 μm and the length of each segment was 800 μm.

     
  3. (3)

    Thermal reflow: The semi-cylindrical structures were constructed by the implementation of a thermal reflow process at 140°C for 20 min.

     
  4. (4)

    Titanium and Aluminum sputtering: A 30 nm thick thin film of titanium and a 10–15 μm thick layer of aluminum were sputtered in sequence onto the semi-cylindrical microvessel network. The titanium thin film served as an adhesive layer.

     
  5. (5)

    Anodization: Anodization of aluminum foil in a 0.3 M oxalic acid solution under a 90 V applied voltage at 0°C for 2 h was then conducted. For comparison purposes, an additional anodization process using 0.3 M phosphoric acid was conducted under a 120 V applied voltage at 0°C for 4 h.

     

2.1.2 PLGA solution preparation

The PLGA solution was prepared by dissolving 85/15 POLY, IV:1.6–1.99 (dl/g), Mw:350000–500000 (Da) (Bio Invigor Corp., Taiwan) in acetone at a w/w ratio of 1:4. The mixture was then stirred with a magneto agitator for 60 min at 60°C. The PLGA solution was then shaken in an ultrasonic shaker for 15 min to remove bubbles created during mixing.

2.2 Fabrication of the microvessel scaffold with nano-patterned inner-walls

2.2.1 Micromolding the PLGA substrates

The procedures used to fabricate the PLGA substrates are illustrated in Fig. 3. The PLGA solution was used to take a casting from the nano-porous semi-cylindrical photoresist mold. The mold was first placed in a vessel after which the solution was poured over the top. The system was left at room temperature for 24 h to allow the acetone to gradually evaporate and the polymer to solidify. The vessel holding the PLGA membrane and the photoresist mold was then immersed in cold water for 2–3 min. The difference between the coefficients of thermal expansion of the materials allowed the micromolded PLGA membrane to be easily stripped off the photoresist mold.
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Fig. 3

Procedures used to fabricate the PLGA substrate

2.2.2 Alignment and bonding

Before bonding, the PLGA membranes were immersed in acetone, cleaned by ultrasonic oscillation, and then rinsed in DI water. An ICP (Cirie-100) treatment was applied to modify the surface properties of the PLGA membrane so that the adhesive force at the sides of the membranes greatly increased. The process parameters for the ICP treatment were: pressure = 5 × 10−3 torr; temperature = 150°C; oxygen = 20 sccm, power = 500 W; processing time = 6 sec. After conducting the ICP treatment, the two PLGA membranes were carefully aligned and brought into contact. They immediately bonded tightly due to spontaneous adhesion of the material. A mask aligner (OAI/500) was used to make certain of the alignment.

2.3 Cell seeding

The microvessels were grown with bovine endothelial cells (BEC). Semi-dynamic seeding which allows the cultivation medium to be exchanged periodically and thus reduces the probability of virus infection was used to culture the BECs in the nano-patterned PLGA scaffold. The semi-dynamic seeding is schematically illustrated in Fig. 4. Instead of using a syringe pump, circulation of the cultivation medium is carried out by periodically injecting fresh medium into and sucking aging medium out from the scaffold. A circulation frequency of 2–3 times/day produced seeding outcomes similar to those of dynamic seeding. Conditions of 37°C, 5% CO2, and a relative humidity of 95% were maintained in the incubator (Model 5410, Napco).
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Fig. 4

Semi-dynamic seeding

The cells were stained with Calcein acetoxymethylester (Calcein-AM) to make monitoring easier. The labeling procedure was as follows: Prepare 1 mM Calcein-AM solution with DMSO and dilute at a ratio of 1–50 µM Calcein-AM solution with PBS. An aliquot of Calcein-AM solution with a volume equal to 1/10 of the volume of cell culture medium was added to the well containing the cells in the culture medium. The labeled cell suspension was injected into the PLGA scaffold and the cells allowed to incubate. Observations of the cells were carried out using a fluorescence microscope with 490 nm excitation and 515 nm emission filters.

3 Results and discussions

An optical microscopic (OM) image of the photoresist microstructure after development is shown in Fig. 5. The processing parameters had to be carefully selected to avoid the under-development and/or over-development which would degrade the accuracy of the scaffold.
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Fig. 5

OM image of the photoresist microstructures after development

The images of the semi-cylindrical microchannel structure after completion of the thermal reflow process are shown in Fig. 6. Cross-sectional SEM image, such as that in Fig. 6(b), was obtained by a 3D confocal microscope (NanoFocus AG). The color scale indicates the height measured from the reference point. The height of the semi-cylindrical photoresist mold is about 55 μm.
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Fig. 6

Semi-cylindrical microchannel structure after thermal reflowing: (a) top view by OM; (b) cross-sectional 3D micrograph by confocal microscopy (NanoFocus AG)

An OM image of the aluminum thin film deposited on the semi-cylindrical microchannel structure is shown in Fig. 7. The semi-cylindrical microchannels retained almost the same shapes as the semi-cylindrical microchannel structure patterned in the photoresist.
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Fig. 7

OM image of the aluminum thin film showing the semi-cylindrical microchannel structure after the deposition process

The CCD image of the semi-cylindrical photoresist mold after anodization and the SEM images of the nano-pores on its surface are displayed in Fig. 8. It can be seen that anodic oxidation could be successfully executed on the semi-spherical thin film of aluminum. The pore size of the nanopores on the oxalic acid etched replica mold was around 30–40 nm, while on the phosphoric acid replica mode it was about 100 nm. The replica mode was then used for micromoding the nano-patterned PLGA scaffolds. The diameters of the nano-pores are controllable between 10 nm and 200 nm, depending on the electrolytes (sulfuric acid, oxalic acid, phosphoric acid) used for anodization and the applied voltage. Anodic aluminum oxide anodized in sulfuric acid has smaller pore-size, while it has larger pore-size using phosphoric acid (Li et al. 1998; Li et al. 1999). In our earlier works (Wang et al. 2009), plane nanostructured scaffolds of PLGA with their surface roughness ranging from 25 nm to 76 nm were fabricated. It was suggested that a nanostructured PLGA membrane with smaller surface roughness could be more contributive to the BEC growth. Therefore, two nanostructured scaffolds of microvessel, one having small surface roughness while the other having large surface roughness, using oxalic acid and phosphoric acid as electrolytes respectively were prepared on the purpose of comparisons.
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Fig. 8

Images of the semi-cylindrical photoresist replica mold after anodization; (a) Top view, (b) SEM image of the nano-pores etched by oxalic acid, (c) SEM image of the nano-pores etched by phosphoric acid

The atomic force microscopic (AFM) micrographs of the nano-patterned PLGA substrates micromolded from the molds shown in Figs. 8 are presented in Fig. 9. The surface roughness of the PLGA substrates was 20 nm and 80 nm for the oxalic acid etched and phosphoric acid etched molds, respectively.
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Fig. 9

AFM images of the nano-patterned semi-cylindrical PLGA microchannel structures after micromolding; (a) micromoded by an oxalic acid etched replica mold, (b) micromoded by a phosphoric acid etched replica mold

An OM image of a microchannel cross-section in the nano-patterned PLGA microvessel scaffold is displayed in Fig. 10. To ensure that the seals between the top and bottom PLGA substrates were robust and leakproof, a syringe pump was used to continuously circulate the cultivation medium inside the scaffold for several minutes.
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Fig. 10

OM image of a nano-patterned PLGA circular microchannel

The wall of a real capillary is composed of only a single layer of squamous cells, called the endothelium, which is rolled to produce a tube. This cell wall enables the exchange of water, oxygen, carbon dioxide, and nutrients (driven by osmotic and hydrostatic gradients) between the blood and the surrounding tissues. The purpose of cell seeding is to allow endothelial cells to grow inside the microvessel scaffold and the lumens to take shape.

The PLGA scaffold turned milky and completely opaque within 24 h after cell seeding due to absorption of the cultivation medium and the resultant hydrolysis (Paragkumar N et al. 2006; Croll et al. 2004). This made conventional optical microscopy an ineffective choice for monitoring the cell seeding progress. Calcein-AM has been widely in microscopy and fluorometry to provide both morphological and functional information of viable cells. It is one of the most suitable fluorescent dyes for labeling viable cells because of its low cytotoxicity and high hydrophobicity (Jonsson et al. 1996; Legrand et al. 1998; Uggeri et al. 2004). It is not only an indicator of presence/absence of cells but also the viability of cells. It does not inhibit cellular functions such as the proliferation or chemotaxis of lymphocytes.

Figures 11 and 12 show fluorescence microscopic images (Eclipse 80i, Nikon) of the Calcein-AM labeled BECs recorded after 3 and 7 days of seeding, respectively. The cells adhered well to the walls and grew in an orderly fashion along the nano-patterned microchannels. For the results of the 3 days’ seeding shown in Fig. 11, the differences in cell growth were not so apparent. However, it can be observed from Fig. 12 that when the surface roughness of the PLGA scaffold was 20 nm, BEC cell growth was more separated, but when the scaffold had a surface roughness of 80 nm the cell growth was more aggregated, In other words, BECs grew better in a nano-patterned microvessel scaffold with less surface roughness. In our earlier works (Wang et al. 2009), it was observed that the nanostructured PLGA membrane could increase the cell growing rate, especially for the membrane with smaller surface roughness. Although the framework of the scaffold in this study is different from the early works, the results of this study agree with those of the earlier works to a certain degree.
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Fig. 11

Fluorescence microscopic images if Calcein-AM labeled BECs after 3 days of semi-dynamic seeding

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Fig. 12

Fluorescence microscopic images of Calcein-AM labeled BECs after 7 days of semi-dynamic seeding

It was also observed that the cells inside the nanostructured microchannels appeared more rounded when compared with the cells cultured on a flat PLGA surface. It is presumed that the different geometry of the substrates and the semi-dynamic seeding that flushed the cells by the culture medium 2–3 times daily. Although the cells inside the microchannels did not completely cover the entire surface, we noted that about 90% of the cells showed fluorescence, i.e. remained healthy.

For comparison, the fluorescence microscopy images for the smooth surface scaffold recorded after 7 days of seeding are shown in Fig. 13. It can be observed that the cells grew more separately in a nano-patterned microvessel scaffold than they did in a smooth surface scaffold. Since the BECs could only be injected to the scaffolds through the inlet, the distribution of the cells inside the scaffold might not be so uniform. Therefore, the difference in the cell count for the nanostructured scaffold and the smooth scaffold may vary with the area of focus.
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Fig. 13

Fluorescence microscopy images for the smooth surface scaffold recorded after 7 days of semi-dynamic seeding

Because the semi-dynamic seeding rather than the dynamic seeding was used to culture the BECs inside the microvessel scaffold, the adhesiveness of cells and resistance to flow at various shear stresses are not available.

It has been suggested that focal adhesion formation and cytoskeletal structure of cultured cells could be affected by the local differences in interfacial forces, especially for nanoscale topographies (Dalby 2005). Lampin et al. (1997) proposed that the roughness of PMMA surfaces might induce the sub-confluent cells to excrete extra-cellular proteins which allowed better catching of cells to the substrate. Kieswetter et al. (1996) reported that the production of cytokine and growth factor of cells could be modulated by the surface roughness of titanium. Although the material and cell type employed in this study were different from those of the earlier works, the aforementioned factors might still be attributed to the enhancement of BEC growth. However, the detail mechanisms for the adhesion and growth enhancement due to surface roughness still need further study.

4 Conclusion

In this study, we combined the anodic aluminum oxide technique and the photoresist thermal reflow method to fabricate PLGA microvessel scaffolds with nanostructured inner walls. To the best of our knowledge, it is the first time that a systematic approach for the fabrication of microvessel scaffolds having nanostructured inner walls is presented. The cell seeding results demonstrated that the BECs grew more separately in a nano-patterned microvessel scaffold than they did in a smooth surface scaffold. Although the progress of better cell seeding was achieved in this research, the formation of a real microvessel network has not yet been seen. Our future works will concentrate on the development of microvessel scaffolds at a size scale and shape that mimics actual vasculature and the seeding of endothelial cells of human microvessels to realize a real microvessel network.

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