The fabrication of PLGA microvessel scaffolds with nano-patterned inner walls
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- Wang, G., Lin, Y. & Hsu, S. Biomed Microdevices (2010) 12: 841. doi:10.1007/s10544-010-9438-x
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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.
KeywordsMicrovessel scaffoldPLGANanostructureCircular microchannelsBovine endothelial cells
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
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
- (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.
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.
Thermal reflow: The semi-cylindrical structures were constructed by the implementation of a thermal reflow process at 140°C for 20 min.
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.
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
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 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
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.
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.
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.
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.