Bio-MEMS fabricated artificial capillaries for tissue engineering
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- Wang, G.J., Chen, C.L., Hsu, S.H. et al. Microsyst Technol (2005) 12: 120. doi:10.1007/s00542-005-0017-7
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In this report, we focus on the microfabrication and cell seeding issues of artificial blood capillaries for tissue engineering. Two different fabrication methods (stainless steel electroforming and silicon electroforming) and a number of materials (PC, Polycarbonate and biocompatible material PLGA, poly lactide-co-glycolides) are implemented to build the vascular network. The vascular network is then used as the scaffold to cultivate the bovine endothelial cell (BEC). During the period of cell cultivation, oxygen and nutrient need to be continuously delivered by a circular pressurizing system. In cell culture, encouraging results are obtained through the dynamical seeding of the BEC on the scaffolds. A systematic cell culture process has been developed after repeated experiments. Successful seeding efficiencies are obtained by using the developed systematic cell culture process.
Most of the vital organs are short of the ability of regeneration. Organ transplant is the only solution to the loss or failure of a vital organ. Shortage of donor organs makes tissue engineering a promising solution to organ transplantation.
Tissue engineering is a new field in science, medicine, and engineering in which bio-artificial organs and tissues are built and then implanted into a live body to repair or increase the functions of the vital organs. The concept of tissue engineering is to cultivate cells in a biodegradable scaffold in which the cells grow to regenerate new organs such as skin, cornea, born, and cartilage. One of the major challenges of the tissue engineering is the lack of intrinsic blood vessels to transport the nutrient and metabolite. Once the tissue is larger then 1–2 mm, the cultivating cells will shrivel due to the lack of metabolic nutrient and air. It is thus desired to provide the artificial tissues with artificial blood vessels or capillaries.
In general, growth accelerant for the cells is spread on the biodegradable scaffold such that the cells can be catalyzed to regenerate blood vessels. However, blood capillaries with diameter in μm are difficult to be constructed by conventional approaches. King et al. (2002) and Borenstein et al. (2002) were the pioneers and primarily microfabricated capillary scaffold on the PolyDiMethylSiloxane (PDMS) and seeded with endothelial cells to form artificial capillaries. Since the PDMS is transparent, the cell cultivating processes can be observed. However, the scaffolds of the vessel network have to be made of a biodegradable material such that the endothelial cells can be successfully seeding and implanted into the body of animals.
In the past few years, bioMEMS technology such as bulk micromachining (Kovacs et al. 1998; Pan et al. 2002), LIGA processing (Chang and Kim 2000; Marques et al. 1997) hot embossing (Jeon and Chiu 2002; Becker and Heim 1999), and excimer laser cutting (Hui and Qin 2002; Kancharla and Chen 2002) have been successfully used to build microfluidic structures and devices such as the microvalve, the micropump, the microreactor, and the biochip in the range of submicron. Many useful biomedical devices have been developed, e.g. biochip and lab-on-a-chip (Madou et al. 2001; Martin et al. 2000; Jiang et al. 2000), biosensor (Polla et al. 2000; Alvarez et al. 2003), electrotactile display (Tang and Beebe 2003). It seems feasible to apply the BioMEMS technology to the manufacturing of the capillary network.
In this article, we focus on the development of the microfabrication method for biocompatible capillaries which are as small as the real capillaries and capable of being observed the cell seeding processes. The microfabrication process of the biocompatible PLGA scaffolds is the core technique of this new developing method. The procedures to micromachine the PLGA scaffolds are: (1) lithograph the silicon wafer or stainless steel substrate (2) process the electroforming to make the mold (3) spin coat the liquid PLGA that is dissolved by certain solvent on the mold (4) use O2 plasma to join the PLGA and the glass together.
2 Design and Fabrication of the Vascular Network
2.1 Vascular Network Design
2.2 Vascular Network Fabrication
The fabrication processes consist of mode fabrication, replica molding, and bonding three steps. For comparisons, two different mode fabrication methods and materials are implemented to build the vascular network.
2.2.1 Replica Mode Fabrication
(1) Stainless steel electroforming method
Stainless steel substrate possesses advantages such as high strength, high corrosion resistance, and can be directly used as conducting layer. Once the thickness of the photoresist is higher than that of the electroforming layer, the metal depositing rate can be controlled through the current density such that the desired electroforming thickness can be precisely met. The drawback is that the adhesion between the substrate and the electroforming layer is not strong enough when the surface of the substrate has not been well cleaned.
(2) Silicon electroforming method
The main advantage of the silicon electroforming method is that the semiconductor fabrication techniques can be easily adopted. However, residual stress that results from the relatively larger volume of the electroforming layer can be a serious problem. Nickel that has less hardness can be used to reduce the residual stress.
2.2.2 Hot Embossing and Bonding
In this research, the high penetrability PC (Polycarbonate) and the biodegradable PLGA (poly lactide-co-glycolides) polymer materials are selected as the substrates. The PC based vascular network is built to enable the cultivating processes to be observed.
(1) PC substrate
During the hot embossing, there are four control variables: preheating, hot embossing temperature, loading pressure, and embossing period are chosen to investigate their influences on the embossing results. It is found from experiments that preheating is crucial to the embossing quality. Figure 7 indicates the embossing result without preheating. The possible reason is that the PC substrate deformed under external loading at room temperature. It is also observed that a shorter embossing time (less than 3 min) results in unwanted microfluidic channels (Fig. 8). However, embossing temperature and loading pressure are the two key factors to the embossing quality.
In our experience, the following parameter set can be implemented to produce better vascular network patterns such as shown in Fig. 9; preheating time = 5 min, embossing temperature = 135°C, loading pressure = 4.5 Mpa, embossing time = 6–10 min. Figure 10 illustrates the surface profile of the vascular network measured by surface roughness metrology.
For the bonding of PC layers, bonding pressure and temperature are the main control variables. Figure 11 shows the bonding results of insufficient temperature and pressure. Figure 12 illustrates the effect of over-heating.
In our experiments, serviceable bonding as shown in Fig. 13 can be carried out by adequate parameter settings such as: preheating time = 5 min, bonding temperature = 137°C, bonding pressure = 5.6 Mpa, bonding time = 6–8 min.
(2) PLGA Substrate
PLGA substrate preparation: Dissolve the PLGA 50/50 (Birmingham Polymers, U.S., Mw ≈ 80,000) into Dioxane (TEDIA); Heat the solution up to 60°C and stir simultaneously to become a 20% PLGA solution.
PVA spin coating (for PLGA demolding): Make the PVA solution with 1% concentration; Spin coat the PVA film on the replica mold.
PLGA spin coating: Spin coat the PLGA film to cover the PVA film; Exhaust at room temperature for 24 hours to evaporate the solvent.
Demolding: Dip the replica mold into DI water for a few hours to hydrolyze the PVA film; Demold to obtain the PLGA based scaffolds.
During cultivation, the PLGA scaffolds become opaque after being immersed in the culture solution for long-term culture. In this research, the PLGA substrate is bonded with glass plate by O2 plasma such that the cultivating processes can be observed and recorded.
3 Cell cultivation
In this work, the bovine endothelial cells (BEC) are adopted to seed on the vascular scaffolds. However, dynamic seeding with circulating flow is much more complicated than simple static seeding on cultivation containers. To gain a deeper understanding about the dynamic seeding on the vascular network, preliminary experiments using a straight microchannel with 20×150 μm2 are conducted. The experimental apparatus is schematically illustrated in Fig. 14.
3.1 Biocompatibility experiments
3.2 Static Seeding
Sterilization: Firstly sterilize the scaffolds and devices by deep UV-light. Followed by a second sterilization by 70% ethanol that is injected through a peristaltic pump. The residual ethanol is then replaced by the buffer solution PBS.
CBD-RGD: Inject the RGD solution into the microchannel; Seal both outlets for 30 min to allow enough reacting time.
Cell suspension preparation: Prepare cell suspension with concentration 106–107 cells/ml.
Static seeding: Pump cell suspension into microchannel; Static seed inside the cultivating container under the conditions of 37°C, 5 CO2, relative humidity 95%.
3.3 Dynamic Seeding
The apparatus for dynamic seeding is shown in Fig. 14, in which the culture medium is continuously circulated by the peristaltic pump so that the environment of a real artery can be imitated. Since the peristaltic pump is set outside the culture container, little crack on the pipe junction may induce serious infection to the seeding cells. It should be carefully avoided.
The main goal of this research is to use the BioMEMS technique to build artificial blood capillaries on both the PC (Polycarbonate) and biocompatible material such as PLGA (poly(lactide-co-glycolides)). Firstly, a vascular network is constructed on both materials. The micro-channel network is then used as the scaffold to cultivate bovine endothelial cells (BECs). The artificial blood capillaries are finally constructed after the endothelial cells grow up and the scaffold is decomposed. During the period of cell cultivation, oxygen and nutrients need to be continuously administered by a circular pressurizing system.
In cell culture, encouraging results are obtained through the dynamical seeding of the BEC on the PC based scaffolds. A systematic cell culture process has been developed after repeated experiments. Successful seeding efficiencies are obtained by using the developed systematic cell culture process.
The authors would like to address their gratitude to the National Science Council of Taiwan for financial support under grant NSC-91-2212-E-005-012. The work was conducted in the Center of Tissue Engineering and Stem Cells Research (TESC) at the National Chung-Hsing University, Taiwan. The center is funded by National Health Research Institutes (NHRI).