We would like to acknowledge the Science and Technology Planning Project of the Guangdong Province of China (2016B070701007) for supporting this work.

1. Degradable PGA Scaffold based Tissue-engineered Vessels Culture
<ol>
	<li>PGA Scaffold Fabrication
	<ol>
		<li>Sew PGA mesh (19 mm diameter and 1 mm thick) around silicone tubing sterilized by ethylene oxide (17 cm length, 5.0 mm diameter, and 0.3 mm thick) using 5-0 suture.</li>
		<li>Sew polytetrafluoroethylene (ePTFE, 1cm length) with 4-0 suture onto each end of PGA mesh and overlapped by 2 mm.</li>
		<li>Dip PGA scaffolds with the hand in 1 mol/L NaOH for 1 min to adjust the spatial structure of the mesh and soa...

<ol>
	<li>Chan-Park, M. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomimetic+control+of+vascular+smooth+muscle+cell+morphology+and+phenotype+for+functional+tissue-engineered+small-diameter+blood+vessels.">Biomimetic control of vascular smooth muscle cell morphology and phenotype for functional tissue-engineered small-diameter blood vessels.</a> Journal of Biomedical Materials Research Part A. 88, 1104-1121 (2009).</li><li>Ballyns, J. J., Bonassar, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-guided+tissue+engineering.">Image-guided tissue engineering.</a> Journal of Cellular &amp; Molecular Medicine. 13, 1428-1436 (2009).</li><li>Smith, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+imaging+methodologies+for+3D+tissue+engineering.">A comparison of imaging methodologies for 3D tissue engineering.</a> Microscopy Research &amp; Technique. 73, 1123-1133 (2010).</li><li>Chang, W. G., Niklason, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+short+discourse+on+vascular+tissue+engineering.">A short discourse on vascular tissue engineering.</a> NPJ Regenerative Medicine. 2, (2017).</li><li>Appel, A. A., Anastasio, M. A., Larson, J. C., Brey, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+challenges+in+biomaterials+and+tissue+engineering.">Imaging challenges in biomaterials and tissue engineering.</a> Biomaterials. 34, 6615-6630 (2013).</li><li>Rice, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+characterization+of+structure+and+morphology+of+silk+fibroin+biomaterials+using+non-linear+microscopy.">Non-invasive characterization of structure and morphology of silk fibroin biomaterials using non-linear microscopy.</a> Biomaterials. 29, 2015-2024 (2008).</li><li>Niklason, L. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enabling+tools+for+engineering+collagenous+tissues+integrating+bioreactors,+intravital+imaging,+and+biomechanical+modeling.">Enabling tools for engineering collagenous tissues integrating bioreactors, intravital imaging, and biomechanical modeling.</a> Proceedings of the National Academy of Sciences of the United States of America. , 3335-3339 (2010).</li><li>Zheng, K., Rupnick, M. A., Liu, B., Brezinski, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three+Dimensional+OCT+in+the+Engineering+of+Tissue+Constructs:+A+Potentially+Powerful+Tool+for+Assessing+Optimal+Scaffold+Structure.">Three Dimensional OCT in the Engineering of Tissue Constructs: A Potentially Powerful Tool for Assessing Optimal Scaffold Structure.</a> Open Tissue Engineering &amp; Regenerative Medicine Journal. 2, 8-13 (2009).</li><li>Gurjarpadhye, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+and+characterization+of+bioengineered+blood+vessels+within+a+bioreactor+using+free-space+and+catheter-based+OCT.">Imaging and characterization of bioengineered blood vessels within a bioreactor using free-space and catheter-based OCT.</a> Lasers in Surgery and Medicine. 45, 391-400 (2013).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+remodeling+and+structural+characterization+of+degradable+polymer+scaffold-based+tissue-engineered+vascular+grafts+using+optical+coherence+tomography.">In vitro remodeling and structural characterization of degradable polymer scaffold-based tissue-engineered vascular grafts using optical coherence tomography.</a> Cell &amp; Tissue Research. 370, 417-426 (2017).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+natural+history+of+extracellular+matrix+production+in+tissue-engineered+vascular+grafts+during+neovessel+formation.">Characterization of the natural history of extracellular matrix production in tissue-engineered vascular grafts during neovessel formation.</a> Cells Tissues Organs. 195, 60-72 (2012).</li><li>Ye, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+design+conception+and+realization+of+pulsatile+ventricular+assist+devices-from+Spiral-Vortex+pump+to+Luo-Ye+pump.">The design conception and realization of pulsatile ventricular assist devices-from Spiral-Vortex pump to Luo-Ye pump.</a> Chinese Journal of Thoracic and Cardiovascular Surgery. 9, 35-40 (2002).</li><li>Chen, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+optical+coherence+tomography+in+tissue+engineered+blood+vessel+culture+based+on+Luo-Ye+pump.">Application of optical coherence tomography in tissue engineered blood vessel culture based on Luo-Ye pump.</a> Chinese Journal of Thoracic and Cardiovascular Surgery. 31, 687-690 (2015).</li><li>Pickering, J. G., Boughner, D. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+the+age+of+fibrotic+lesions+using+polarized+light+microscopy+and+digital+image+analysis.">Quantitative assessment of the age of fibrotic lesions using polarized light microscopy and digital image analysis.</a> American Journal of Pathology. 138, 1225-1231 (1991).</li><li>Martinho, J. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dependence+of+optical+attenuation+coefficient+and+mechanical+tension+of+irradiated+human+cartilage+measured+by+optical+coherence+tomography.">Dependence of optical attenuation coefficient and mechanical tension of irradiated human cartilage measured by optical coherence tomography.</a> Cell Tissue Bank. 16, 47-53 (2015).</li><li>Poirierquinot, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+1.5-Tesla+magnetic+resonance+imaging+for+tissue-engineered+constructs:+a+noninvasive+tool+to+assess+three-dimensional+scaffold+architecture+and+cell+seeding.">High-resolution 1.5-Tesla magnetic resonance imaging for tissue-engineered constructs: a noninvasive tool to assess three-dimensional scaffold architecture and cell seeding.</a> Tissue Engineering Part C Methods. 16, 185-200 (2010).</li><li>Naito, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+burst+pressure:+initial+evaluation+of+the+natural+history+of+the+biaxial+mechanical+properties+of+tissue-engineered+vascular+grafts+in+the+venous+circulation+using+a+murine+model.">Beyond burst pressure: initial evaluation of the natural history of the biaxial mechanical properties of tissue-engineered vascular grafts in the venous circulation using a murine model.</a> Tissue Engineering Part A. 20, 346-355 (2014).</li><li>Smart, N., Dube, K. N., Riley, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coronary+vessel+development+and+insight+towards+neovascular+therapy.">Coronary vessel development and insight towards neovascular therapy.</a> International Journal of Clinical and Experimental Pathology. 90, 262-283 (2009).</li></ol>

To generate engineered vessels with structural and mechanical properties similar to those of native blood vessels can lead to shorten the time for clinical use and is the ultimate goal of vascular engineering. Optical imaging techniques permit the visualization of tissue engineered vascular specific components, which cannot monitor individual constructs throughout culture and exposure grafts to a culture environment without compromising sterility7. In this article, the culture chamber is separated...

Tissue-engineered blood vessels (TEBVs) is of the most promising material as an ideal vascular graft1. In order to develop grafts to be clinically useful with similar structural and functional properties as native vessels, multiple techniques have been designed to maintain vascular function2,3. Although there have been engineered vessels with acceptable patency rates during implantation and in Phase III clinical study4, long-term culture and high cost also show the necessity of monitoring the development of TEBVs. Understanding of extracellular matrix(ECM) growth, remodeling, and adaptation processes in TEBVs in the biomimetic chemo-mechanical environment can provide crucial information for the development of vascular tissue engineering.
The ideal strategy to track the development of small-diameter engineered vessels5 should be nondestructive, sterile, longitudinal, three-dimensional and quantitative. TEBVs under different culture conditions could be assessed by this imaging modality, even including changes before and after vascular transplantation. Strategies to describe features of living engineered vessels are needed. Optical imaging techniques allow visualization and quantification of tissue deposition and biomaterials. Other advantages are the possibility to enable deep-tissue and label-free imaging with high resolution6,7. However, image-specific molecules and less easily accessible optical equipment for real-time monitoring is a significant practical obstacle, which has limited the extensive application of nonlinear optical microscopy. Optical coherence tomography (OCT) is an optical approach with intravascular imaging modality as a widely-used clinical tool to guide cardiac interventional therapy8. In the literature the method of OCT was reported as a way to assess the wall thickness of TEBVs9,10, coupled with affirmative imaging modalities for vascular tissue engineering research. Whereas, the dynamics of engineered vascular growth and remodeling was not observed.
In this manuscript, we detail the preparation of biodegradable polymeric scaffold-based TEBVs for four weeks culture. Human umbilical arteries vascular smooth muscle cells (HUASMCs) are expanded and seeded into a porous degradable polyglycolic acid (PGA) scaffolds in the bioreactor. Biodegradable polymers play the role in a temporary substrate for tissue engineering and have a certain degradation rate11. In order to ensure an appropriate match between scaffold degradation and neo-tissue formation, ECM and PGA scaffolds are crucial factors for effective vascular remodeling. The perfusion system simulates the biomechanical microenvironment of native vessels and maintains a consistent deformation under pressure stimulation. 
The aim of the presented protocol is to describe a relatively simple and nondestructive strategy for TEBVs imaging and long-term monitoring of culture. This protocol can be utilized for visualization of morphological changes and thickness measurements of engineered vessels under different culture conditions. Additionally, the analyses of polymer-based materials degradation in the tissue engineering scaffolds can be performed for the identification. By combining methods of scanning electron microscopic(SEM) and polarized microscope used in this protocol, correlation and quantification of extracellular matrix distribution and PGA degradation can be made, which can facilitate assessing scaffold degradation combined with OCT imaging.

<table border="0" cellpadding="0" cellspacing="0" style="width:797px;" width="797">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PGA mesh</td>
			<td style="width:265px;">Synthecon</td>
			<td style="width:149px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">silicone tube</td>
			<td style="width:265px;">Cole Parmer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">connector</td>
			<td style="width:265px;">Cole Parmer</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:47px;width:216px;">intravascular OCT system</td>
			<td style="width:265px;">St. Jude Medical, Inc</td>
			<td style="width:149px;">ILUMIEN&#8482; OPTIS&#8482; SYSTEM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">scanning electron microscopic</td>
			<td style="width:265px;">Philips&#160;</td>
			<td style="width:149px;">FEI Philips XL-30</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">polarized microscope</td>
			<td style="width:265px;">Olympus</td>
			<td style="width:149px;">Olympus BX51</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:31px;width:216px;">sutures</td>
			<td style="width:265px;">Johnson &#38; Johnson</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:216px;">pulsatile pump</td>
			<td style="width:265px;">Guangdong Cardiovascular Institute</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">LightLab Imaging software</td>
			<td style="width:265px;">St. Jude Medical, Inc</td>
			<td style="width:149px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

nondestructive monitoring of degradable scaffold-based tissue-engineered blood vessel development using optical coherence tomography

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial bypass. Characterization of the growth dynamics and remodeling process of degradable polymer scaffold-based tissue-engineered blood vessels (TEBVs) with pulsatile stimulation is crucial for vascular tissue engineering. Optical imaging techniques stand out as powerful tools for monitoring vascularization of engineered tissue enabling high-resolution imaging in real-time culture. This paper demonstrates a nondestructive and fast real-time imaging strategy to monitor the growth and remodeling of TEBVs in long-term culture by using optical coherence tomography (OCT). Geometric morphology is evaluated, including vascular remodeling process, wall thickness, and comparison of TEBV thickness in different culture time points and presence of pulsatile stimulation. Finally, OCT provides practical possibilities for real-time observation of the degradation of polymer in the reconstructing tissues under pulsatile stimulation or not and in each vessel segment, by compared with the assessment of polymer degradation using scanning electron microscopic(SEM) and polarized microscope.

The three-dimensional culture system consisted of a culture chamber in the bioreactor and the perfusion system with a closed fluid cycle10,13 (Figure 1). The OCT imaging catheter was inserted into the distal end of the Y-junction and pulled back in the silicone tube for imaging. OCT imaging was first used to delineate the structural characterization of biodegradable polymeric scaffold-based TEBVs duri...

Watch this Scientific Journal Video about Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography at JoVE.com

Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography

A step by step protocol for nondestructive and long-period monitoring the process of vascular remodeling and scaffold degradation in real-time culture of biodegradable polymeric scaffold-based tissue-engineered blood vessels with pulsatile stimulation using optical coherence tomography is described here.

Engineered vascular grafts with structural and mechanical properties similar to natural blood vessels are expected to meet the growing demand for arterial ...

This method can help answer key questions in the vascular tissue-engineering field such as monitoring the dynamics of engineer vascular growth and remodeling during long-term culture. The main advantage of using optical coherence tomography, or OCT, is that it is a readily available, realtime and nondestructive imaging strategy to characterize the structural features and remodeling process of engineered vessels. Demonstrating the procedure will also be Shangmin Liu, and Wentao Ma, technicians from my laboratory.To begin this procedure, fabricate the PGA scaffold as described in the text protocol Dip PGA scaffolds in one mol-per-liter sodium hydroxide for one minute, to adjust the spacial structure of the mesh. Then, soak the scaffolds in tissue-culture-grade water three times for two minutes each. Each time, gently pat dry the scaffold with a tissue paper.Then, dry up the scaffolds in a hood with a blower for one hour. To assemble the bioreactor and the y-junction for OCT imaging, first, soak the self-developed glass cylindrical bioreactor, PGA scaffolds, silicone tube, bio-compatible tubes, connectors, stir bar, and equipment for assembly in a 95 percent ethanol tank for two hours. Pull the PGA scaffold through the side arms of the bioreactor connected to one side with a connector, as well as to another side with the y-junction used to deliver the OCT guide wire.Assemble another PGA scaffold in the bioreactor in the same way. Fit the polytetrafluoroethylene to the bioreactor lips by tightening it with 4-0 sutures. Put the bioreactor in the ethanol tank again for one hour before drying it overnight in a hood with the blower on.Now, isolate human umbilical artery smooth muscle cells from human umbilical arteries by standard explant techniques In the cause of saving the cells into PGA scaffolds, avoid any dripping of cell suspension on the bottom of bioreactor. Also, cover the bioreactor with silicone-stopper lid as soon as possible after cell seating to prevent contamination. Expand and maintain the cells in smooth muscle growth medium.Seed the human umbilical artery smooth muscle cells at a concentration of 5 million cells-per-milliliter in the above culture medium onto the PGA scaffolds. After placing a sir bar in the bioreactor, cover it the silicone stopper lid that has one feeding tube and three short tubing segments inserted for gas exchange. Attach PTFE 0.22 micron filters to each air change tube, and attach one heparin cap to the feeding tube.Adjust the stir bar with a stirring speed of 13 rounds-per-minute. Then, assemble the glass bioreactor, silicone stopper lid, and PGA scaffold into the culture system. Allow the cells to adhere for 45 minutes by leaning the bioreactor every 15 minutes with a stand to the left and to the right.Now, connect the LOO-OH-YEE pump, phosphate-buffered saline bag, and driver with the biocompatible tubes in order to create the profusion system. Open the driver to fill the tubes with PBS. Place the overall bioreactor in a humidified incubator with 5 percent CO2 at 37 degrees Celsius.Fill the culture chamber with 450 milliliters of the culture medium. Grow the seeded scaffolds under static culture for one week, changing the culture medium every three to four days by aspirating half of the old medium through the feeding tube and refilling the reactor with an equivalent amount of fresh culture medium. To prepare the profusion system for OCT imaging, pump fluids in the PBS bag to circulate them through the biocompatible tubes and back to the bag.Open the power of the driver and regulate the pump setting with a frequency of 60 beats-per-minute and an output systolic pressure of 120 millimeters of mercury. Adjust the mechanical parameters according to the needs of the tissue-engineering vascular culture. Click the run button to make the profusion system work.Provide the fix pulsatile simulation to the vessels for three weeks by iteratively pressurizing the biocompatible tubes after one week of static culture. Use a light source to ensure the axial resolution of ten to twenty microns and the image depth of one to two millimeters to identify the structure of the tissue-engineered blood vessels, or TEVBs, based on the frequency domain OCT intravascular imaging system. Turn on the power switch and open the image capture software Connect the fiber-optic imaging catheter to the drive motor and optical controller, with the catheter automatic retreat function.Set the parameters of the image acquisition rate to ten frames-per-second, with an automatic pullback speed of ten millimeters-per-second. Now, attach the imaging catheter to the y-junction via the heparin cap with an 18-gauge needle. Place the catheter into the silicone tube and identify the structure tightness of the PGA mesh before loading the PGA scaffold onto the bioreactor.Now, place the catheter tip over the region of interest. Adjust the pullback device and check for the image quality. Acquire images at one, four, seven, ten, 14, 17, 21, and 28 days in culture for each individual TEBV.Save these images sequentially with a realtime observation of the TEBV microstructure including surface morphology, internal structure, and composition. Repeat the measurement three times to get a reliable measurement of the engineered vessels each time. Capture a series of images throughout the testing using the image capture software.To use image analysis software to measure the tissue-engineered blood vessel wall thickness, first select the image to be analyzed. Then, click the tracking tool to automatically identify the inner side of the tissue-engineered blood vessel with the software and manually sketch the outer side. A diagram of thickness will appear on the screen.Repeat the measurement for five times to get a reliable measurement of the constructs. Consider using two independent investigators obligated to the obtain information. Open the silicone stopper lid placed over the bioreactor when the culture is finished, and discard the culture medium.Loosen the polytetrafluoroethylene from the bioreactor lips and cut the silicone tubes from the outer side of the polytetrafluoroethylene with scissors. Harvest the TEBVs from the bioreactor, and cut them into sections for a scanning-electron microscopy examination. Pull out the supporting silicone tube, and fix the sections with 4 percent power formaldehyde.Perform routine histological staining with masson's trichrome and sirious red to examine the morphology of collagen and PGA. To assess the PGA content and collagen component, observe histologic samples with sirious red staining through a polarized microscope. OCT images are compared with histopathological finds of TEBVs after four weeks of culture.The OCT image shows compact microstructure and specific components compared with histological assessment. The culture medium, the silicone tube, the TEBV, and the PGA fragment are visible. Masson's trichrome staining demonstrates collagen fibers distributed in a certain direction, along with PGA remnants in the media layer of the engineered vessels.Sirius red staining reveals PGA remnants and a collagen component by using a polarized microscope. Scanning-electron micrographs of engineered vessels demonstrate a compact microstructure. Meanwhile, this intraluminal imaging modality adopts nondestructive and easy monitoring of TEBVs, including the remodeling process and visual morphology in-situ in long-running culture.As shown here, the remodeling occurs earlier and the morphological changes manifest more obviously in the dynamic group through comparison of TEBV thickness. This technique paves the way for researchers in the field of vascular tissue engineering to explore structure features and the long-term remodeling process of engineered vessels. Clear display of polymeric remnants by polarized microscopy combined with OCT imaging might be useful for assessing scaffold degradation.

nondestructive-monitoring-degradable-scaffold-based-tissue-engineered

Research

JoVE Journal

Bioengineering

5.5K Views.  Guangdong Academy of Medical Sciences, Guangdong General Hospital. This method can help answer key questions in the vascular tissue-engineering field such as monitoring the dynamics of engineer vascular growth and remodeling during long-term culture. The main advantage of using optical coherence tomography, or OCT, is that it is a readily available, realtime and nondestructive imaging strategy to characterize the structural features and remodeling process of engineered vessels. Demonstrating the procedure will also be Shangmin Liu, and Wentao Ma, technicians...