Chang Mo Hwang

Microfluidic Chip-Based Fabrication of PLGA Microfiber Scaffolds for Tissue Engineering

In this paper, we have developed a method to produce poly(lactic- co-glycolic acid) (PLGA) microfibers within a microfluidic chip for the generation of 3D tissue engineering scaffolds. The synthesis of PLGA fibers was achieved by using a polydimethylsiloxane (PDMS)-based microfluidic spinning device in which linear streams of PLGA dissolved in dimethyl sulfoxide (DMSO) were precipitated in a glycerol-containing water solution. By changing the flow rate of PLGA solution from 1 to 50 microL/min with a sheath flow rate of 250 or 1000 microL/min, fibers were formed with diameters that ranged from 20 to 230 microm. The PLGA fibers were comprised of a dense outer surface and a highly porous interior. To evaluate the applicability of PLGA microfibers generated in this process as a cell culture scaffold, L929 fibroblasts were seeded on the PLGA fibers either as-fabricated or coated with fibronectin. L929 fibroblasts showed no significant difference in proliferation on both PLGA microfibers after 5 days of culture. As a test for application as nerve guide, neural progenitor cells were cultured and the neural axons elongated along the PLGA microfibers. Thus our experiments suggest that microfluidic chip-based PLGA microfiber fabrication may be useful for 3D cell culture tissue engineering applications.

Characterization of low-molecular-weight hyaluronic acid-based hydrogel and differential stem cell responses in the hydrogel microenvironments

Hyaluronic acid is a natural glycosaminoglycan involved in biological processes. Low-molecular-weight hyaluronic acid (10 and 50 kDa)-based hydrogel was synthesized using derivatized hyaluronic acid. Hyaluronic acid was acrylated by two steps: (1) introduction of an amine group using adipic acid dihydrazide, and (2) acrylation by N-acryloxysuccinimide. Injectable hyaluronic acid-based hydrogel was prepared by using acrylated hyaluronic acid and poly(ethylene glycol) tetra-thiols via Michael-type addition reaction. Mechanical properties of the hydrogel were evaluated by varying the molecular weight of acrylated hyaluronic acid (10 and 50 kDa) and the weight percent of hydrogel. Hydrogel based on 50-kDa hyaluronic acid showed the shortest gelation time and the highest complex modulus. Next, human mesenchymal stem cells were cultured in cell-adhesive RGD peptide-immobilized hydrogels together with bone morphogenic protein-2 (BMP-2). Cells cultured in the RGD/BMP-2-incorporated hydrogels showed proliferation rates higher than that of control or RGD-immobilized hydrogels. Real-time RT-PCR showed that the expression of osteoblast marker genes such as CBFalpha1 and alkaline phosphatase was increased in hyaluronic acid-based hydrogel, and the expression level was dependent on the molecular weight of hyaluronic acid, RGD peptide, and BMP-2. This study indicates that low-molecular-weight hyaluronic acid-based hydrogel can be applied to tissue regeneration as differentiation guidance materials of stem cells.

Simultaneous generation of chemical concentration and mechanical shear stress gradients using microfluidic osmotic flow comparable to interstitial flow.

Cells are very sensitive to various microenvironmental cues, including mechanical stress and chemical gradients. Therefore, physiologically relevant models of cells should consider how cells sense and respond to microenvironmental cues. This can be accomplished by using microfluidic systems, in which fluid physics can be realized at a nanoliter scale. Here we describe a simple and versatile method to study the generation of chemical concentration and mechanical shear stress gradients in a single microfluidic chip. Our system uses an osmotic pump that produces very slow (<a few microm/s) and controlled flow, allowing a wide and stable diffusion of specific chemical concentration. We also established a shear stress gradient passively via a circular channel in the interstitial level. For evaluation of the system, we used L929 mouse fibroblast cells and simultaneously exposed them to a mechanical stress gradient and a chemical nutrient gradient. The interstitial shear stress level clearly affected cell alignment, mobility velocity, and attachment. At the same time, cell proliferation reflected nutrient concentration level. Our system, which enables continuous and long-term culture of cells in a combined chemical and mechanical gradient, provides physiologically realistic conditions and will be applicable to studies of cancer metastasis and stem cell differentiation.

The effects of pulsatile flow upon renal tissue perfusion during cardiopulmonary bypass: A comparative study of pulsatile and nonpulsatile flow

This study was conducted to directly compare the effects of pulsatile and nonpulsatile blood flow in the extracorporeal circulation upon renal tissue perfusion by using a tissue perfusion measurement system. A total cardiopulmonary bypass circuit was constructed to accommodate twelve Yorkshire swine, weighing 20∼30 kg. Animals were randomly assigned to group 1 (n = 6, nonpulsatile centrifugal pump) or group 2 (n = 6, pulsatile T-PLS pump). A tissue perfusion measurement probe (Q-Flow 500) was inserted into the renal parenchymal tissue, and the extracorporeal circulation was maintained for an hour at a pump flow rate of 2 L/min after aortic cross-clamping. Tissue perfusion flow in the kidney was measured before bypass and every 10 minutes after bypass. Renal tissue perfusion flow was substantially higher in the pulsatile group throughout bypass (ranging 48.5-64.1 ml/min/100 g in group 1 vs. 51.0-88.1 ml/min/100 g in group 2). The intergroup difference was significant at 30 minutes (47.5 ± 18.3 ml/min/100 g in group 1 vs. 83.4 ± 28.5 ml/min/100 g in group 2; p = 0.026). Pulsatile flow achieves higher levels of tissue perfusion of the kidney during short-term extracorporeal circulation. A further study is required to observe the effects of pulsatile flow upon other vital organs and its long-term significance.

Ice-lithographic fabrication of concave microwells and a microfluidic network

We describe a novel method to produce concave microwells utilizing solid–liquid phase change. This method, named ‘ice-lithography’, does not require any lithographic processes and consists of a few simple steps that yield multiple concave microwells. We demonstrated that the shape and size of the microwells can be controlled by varying substrates and vapor-collection time. Patterned wells with sizes in the range of 10μm to several millimeters in diameter could be produced. Additionally, we fabricated a uniformly aligned concave microwell pattern and a microfluidic network. Ice-lithography has potential biological and biomedical applications in areas such as the fabrication of cell docking devices and microbioreactors as well as the formation of uniformly sized embryoid bodies.

In vitro evaluation of the performance of Korean pulsatile ECLS (T-PLS) using precise quantification of pressure-flow waveforms

The Twin-Pulse Life Support System (T-PLS) is a novel pulsatile extracorporeal life support system developed in Korea. It has been reported that the T-PLS achieves higher levels of tissue perfusion of the kidney during short-term extracorporeal circulation and provides more blood flow to coronary artery than nonpulsatile blood pumps. However, these results lack pulsatility quantifications and thus make it hard to analyze the effects of pulsatility upon hemodynamic performance. We have adopted the concepts of hemodynamic energy, energy equivalent pressure (EEP), and surplus hemodynamic energy (SHE) to evaluate pulsatility performance in the different circuit configurations of the T-PLS and a membrane oxygenator (MO) in vitro. In a mock system, three different circuits were constructed depending on the location of an MO: pump-MO-pump (serial), MO-pumps (parallel A), and pumps-MO (parallel B). In parallel A, a low-resistance MO was used to preserve the pulsatility from the pump. All circuits showed good pulsatility in terms of EEP (serial: 13.2% ± 3.2%, parallel A: 10.0% ± 1.6%, parallel B: 7.00% ± 1.1%; change from aortic pressure to EEP; p < 0.003). The SHE levels were 17,404 ± 3,750 ergs/cm3, 13,170 ± 1,486 ergs/cm3, and 9,192 ± 1,122 ergs/cm3 in each circuit setup (p < 0.001). Although EEP levels were somewhat lower, both parallel types provided higher pump output compared with the serial type (serial: 1.87 ± 0.29 l/min, parallel A: 3.09 ± 0.74 l/min, parallel B: 3.06 ± 0.56 l/min; p < 0.003 except parallel A vs. parallel B, p = 0.90). Conclusively, the precise quantifications of pressure flow waveforms, EEP, and SHE are valuable tools for evaluating pulsatility of the mechanical circulatory devices, and are expected to be used as additional performance indexes of a blood pump. The pulsatility performances are different according to circuit setups. However, the parallel A circuit could achieve higher pump output and generate adequate pulsatility level. Thus, the parallel A circuit is suggested as the optimal configuration in T-PLS applications.

Benchtop fabrication of PDMS microstructures by an unconventional photolithographic method

Poly(dimethylsiloxane) (PDMS) microstructures have been widely used in bio-microelectromechanical systems (bio-MEMS) for various types of analytical, diagnostic and therapeutic applications. However, PDMS-based soft lithographic techniques still use conventional microfabrication processes to generate a master mold, which requires access to clean room facilities and costly equipment. With the increasing use of these systems in various fields, the development of benchtop systems for fabricating microdevices is emerging as an important challenge in their widespread use. Here we demonstrate a simple, low-cost and rapid method to fabricate PDMS microstructures by using micropatterned poly(ethylene glycol) diacrylate (PEGDA) master molds. In this method, PEGDA microstructures were patterned on a glass substrate by photolithography under ambient conditions and by using simple tools. The resulting PEGDA structures were subsequently used to generate PDMS microstructures by standard molding in a reproducible and repeatable manner. The thickness of the PEGDA microstructures was controllable from 15 to 300 µm by using commonly available spacer materials. We also demonstrate the use of this method to fabricate microfluidic channels capable of generating concentration gradients. In addition, we fabricated PEGDA microstructures by photolithography from the light generated from commonly available laminar cell culture hood. These data suggest that this approach could be beneficial for fabricating low-cost PDMS-based microdevices in resource limited settings.

The effects of pulsatile versus non-pulsatile extracorporeal circulation on the pattern of coronary artery blood flow during cardiac arrest.

Background In sudden cardiac arrest, the effective maintenance of coronary artery blood flow is of paramount importance for myocardial preservation as well as cardiac recovery and patient survival. The purpose of this study was to directly compare the effects of pulsatile versus non-pulsatile circulation to coronary artery flow and myocardial preservation in a cardiac arrest condition. Methods A cardiopulmonary bypass circuit was constructed in a ventricular fibrillation model using fourteen Yorkshire swine weighing 25–35 kg each. The animals were randomly assigned to group I (n=7, non-pulsatile centrifugal pump) or group II (n=7, pulsatile T-PLS pump). Extracorporeal circulation was maintained for two hours at a pump flow of 2 L/min. The left anterior descending coronary artery flow was measured with an ultrasonic coronary artery flow measurement system at baseline (before bypass) and at every 20 minutes after bypass. Serologic parameters were collected simultaneously at baseline, 1 hour, and 2 hours after bypass in the systemic arterial and coronary sinus venous blood. The Mann-Whitney U test of STATISTICA 6.0 was used to determine intergroup significances using a p value of < 0.05. Results The resistance index of the coronary artery was lower in group II and the difference was significant at 40 min, 80 min, 100 min and 120 min (p < 0.05). The mean velocity of the coronary artery was higher in group II throughout the study, and the difference was significant from 20 min after starting the pump (p < 0.05). The coronary artery blood flow was higher in group II throughout the study, and the difference was significant from 40 min to 120 min (p < 0.05) except at 80min. Serologic parameters showed no differences between the groups at 1 hour and 2 hours after bypass in the systemic and coronary sinus blood (p=NS). Conclusion In the cardiac arrest condition, pulsatile extracorporeal circulation provides more blood flow, higher flow velocity and less resistance to coronary artery than non-pulsatile circulation.

Ventricular assist device implantation using a right thoracotomy

Most patients needing implantation of a ventricular assist device (VAD) require repeated sternotomy; some after cardiac surgery, and others later for heart transplantation. The purpose of this study was to establish the right thoracotomy technique as an alternative for VAD implantation to reduce repeated sternotomy-related morbidity and mortality. We performed a right thoracotomy in animals, preclinical cadaver fitting tests, and a clinical case. A total of 20 various animals underwent right thoracotomy for implantation of bi-VAD (BVAD, n = 17) and left VAD (LVAD, n = 3). The right chest cavity was entered through the fourth intercostal space with partial resection of the fifth rib. There was no procedure-related morbidity or mortality, except for one calf with right anterior leg paralysis. Preclinical fitting tests were performed on 7 human cadavers to observe the anatomical feasibility of BVAD cannulation from the right side of the heart. In humans, the ascending aorta, interatrial groove, right atrium, and main pulmonary artery were identified as optimal cannula insertion sites for BVAD implantation. A patient with cardiogenic shock underwent a right thoracotomy for implantation of an external LVAD. Cardiac function recovered after 3 weeks, and the device was successfully explanted through a repeat right thoracotomy. In conclusion, a right thoracotomy can be an alternative method to the standard median sternotomy for patients who need repeated sternotomy because of previous cardiac surgery, transplantation at a later date, or those with mediastinal infections.

Development of a closed air loop electropneumatic actuator for driving a pneumatic blood pump.

In this study, we developed a small pneumatic actuator that can be used as an extracorporeal biventricular assist device. It incorporated a bellows-transforming mechanism to generate blood-pumping pressure. The cylindrical unit is 88 +/- 0.1 mm high, has a diameter of 150 +/- 0.1 mm, and weighs 2.4 +/- 0.01 kg. In vitro, maximal outflow at the highest pumping rate (PR) exceeded 8 L/min when two 55 mL blood sacs were used under an afterload pressure of 100 mm Hg. At a pumping rate of 100 beats per minute (bpm), maximal hydraulic efficiency was 9.34% when the unit supported a single ventricle and 13.8% when it supported both ventricles. Moreover, pneumatic efficiencies of the actuator were 17.3% and 33.1% for LVAD and BVAD applications, respectively. The energy equivalent pressure was 62.78 approximately 208.10 mm Hg at a PR of 60 approximately 100 bpm, and the maximal value of dP/dt during systole was 1269 mm Hg/s at a PR of 60 bpm and 979 mm Hg/s at a PR of 100 bpm. When the unit was applied to 15 calves, it stably pumped 3 approximately 4 L/min of blood at 60 bpm, and no mechanical malfunction was experienced over 125 days of operation. We conclude that the presently developed pneumatic actuator can be utilized as an extracorporeal biventricular assist device.