Name: nondestructive monitoring of degradable scaffold-based tissue-engineered blood vessel development using optical coherence tomography
Uploaded: 2018-10-03T14:00:00.000+00:00
Duration: 11 min 12 s
Description: Watch this Scientific Journal Video about Nondestructive Monitoring of Degradable Scaffold-Based Tissue-Engineered Blood Vessel Development Using Optical Coherence Tomography at JoVE.com

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 soak with tissue culture grade water three times for 2 min each. Gently pat dry the scaffold with a tissue paper each time. Then dry up scaffolds in a hood with a blower for 1 h.</li>
	</ol>
	</li>
	<li>Assembly of bioreactor and the Y-junction for OCT imaging
	<ol>
		<li>Soak the self-developed glass cylindrical bioreactor (10 cm diameter and 11.7 cm in height with four lips inside and four side-arms outside the reactor as shown in Figure 1), PGA scaffolds, silicone tube (external diameter 5 mm, thickness 0.3 mm), biocompatible tubes, connectors, stir bar and equipment for assembly in a 95% ethanol tank for 2 h.</li>
		<li>Pull PGA scaffold through side-arms of the bioreactor connected to one side with a connector as well as another side with the Y-junction used to deliver OCT guidewire. Assemble another PGA scaffold in the bioreactor in the same way. Please refer to Figure 1.</li>
		<li>Fit ePTFE to bioreactor lips by tightening with 4-0 sutures.</li>
		<li>Put the bioreactor in the ethanol tank again for 1 h and dry up overnight in hood with blower on.</li>
	</ol>
	</li>
	<li>Seeding of HUASMCs and Static Bioreactor Conditioning
	<ol>
		<li>Isolate HUASMCs from human umbilical arteries by standard explant techniques.</li>
		<li>Expand and maintain cells in smooth muscle cell growth medium composed of DMEM medium, 20% fetal bovine serum, 2.36 mg/mL HEPES, 100 U/mL penicillin G, 50 &#181;g/mL proline, 20 &#181;g/mL alanine, 50 &#181;g/mL glycine, 1.5 &#181;g/mL CuSO4, 50 &#181;g/mL ascorbic acid, 10 ng/mL basic fibroblast growth factor and 10 ng/mL platelet-derived growth factor.</li>
		<li>Seed HUASMCs at a concentration of 5&#215;106 cells/mL in the above culture medium onto the PGA scaffolds.</li>
		<li>Put a stir bar (1.5 cm length) in the bioreactor. Insert one feeding tube (5 mm diameter, 15 cm length) and three short tubing segments (5 mm diameter, 7 cm length) for gas exchange through the silicone stopper lid.</li>
		<li>Attach PTFE 0.22 &#181;m filters to each air change tube and one heparin cap to the feeding tube. Adjust the stir bar with a stirring speed of 13 rounds per minute. Assemble the glass bioreactor, silicone stopper lid and PGA scaffold into the culture system.</li>
		<li>Allow HUASMCs to adhere for 45 min by leaning the bioreactor every 15 min with stand, to the left and right. The reactor ports and joints are all sealed with paraffin film.</li>
		<li>Connect the Luo-Ye pump, PBS bag, the driver with biocompatible tubes as the perfusion system. Open the drive to fill the tubes with PBS.</li>
		<li>Place the overall bioreactor in a humidified incubator with 5% CO2 at 37 &#176;C. Fill the culture chamber with 450 mL of the HUASMCs culture medium.</li>
		<li>Press the stop button and turn off the power of the drive device. Grow the seeded scaffolds under static culture for one week.</li>
		<li>Change the culture medium every 3-4 days by aspirating half of the old medium through the feeding tube and refilling the reactor with an equivalent amount of fresh culture medium.</li>
	</ol>
	</li>
	<li>Preparation of the perfusion system for OCT imaging
	<ol>
		<li>Pump fluids in the PBS bag to circulate through biocompatible tubes and back to the bag.</li>
		<li>Open the power of the driver and regulate pump setting with a frequency of 60 beats per minute and output systolic pressure of 120 mmHg. Adjust the mechanical parameters according to the needs of tissue engineering vascular culture.</li>
		<li>Click the run button to make the perfusion system work. Provide the above fixed pulsatile stimulation to the vessels for 3 weeks by iteratively pressurizing biocompatible tubes10,12 after 1 week of static culture.</li>
	</ol>
	</li>
</ol>
2. Performing Optical Imaging with OCT
<ol>
	<li>Use a light source to ensure the axial resolution of 10-20&#181;m and the image depth of 1-2 mm to identify the structure of TEBV based on the frequency-domain OCT intravascular imaging system9.</li>
	<li>Turn on the power switch and open the image capture software.</li>
	<li>Connect the fiber optic imaging catheter to the drive-motor and optical controller (DOC) with catheter automatic retreat function.</li>
	<li>Set parameters of image acquisition rate to 10 frames per second with an automatic pullback speed of 10 mm/s.</li>
	<li>Attach imaging catheter to Y-junction via heparin cap with a 18G needle.</li>
	<li>Place the catheter into the silicone tube and identify the suture tightness of PGA mesh before loading PGA scaffold onto the bioreactor.</li>
	<li>Place the catheter tip over the region of interest. Adjust the pullback device and check for the image quality8.</li>
	<li>Acquire images at 1, 4, 7, 10, 14, 17, 21, 28 days in culture for each individual TEBV and save sequentially with real time observation of TEBV microstructure, including surface morphology, internal structure and composition.</li>
	<li>Repeat the measurement for 3 times to get reliable measurement of engineered vessels each time. Capture a series of images throughout testing using the image capture software.</li>
</ol>
3. Imaging Analysis
<ol>
	<li>Use image analysis software to measure TEBV wall thickness. Select the image to be analyzed. Click the tracking tool to identify the inner side of TEBV by the software automatically and manually sketch the outer side. A diagram of thickness will appear on the screen.</li>
	<li>Repeat the measurement for 5 times to get reliable measurement of the constructs. The OCT analysis was performed by two independent investigators blinded to the obtained information.</li>
</ol>
4. Harvest of TEBV and Tissue Processing
<ol>
	<li>Open the silicone stopper lid placed over the bioreactor when the culture is finished and discard the culture medium. Loosen ePTFE from bioreactor lips and cut the silicone tubes from the outer side of ePTFE with scissors. Harvest TEBVs from the bioreactor and cut into sections for scanning electron microscopy examination.</li>
	<li>Take out the rest of TEBVs and cut into 4 &#181;m thick sections. Pull out the supporting silicone tube and fix sections with 4% paraformaldehyde. Perform routine histological staining of Masson&#39;s trichrome and Sirius red to examine the morphology of collagen and PGA10,13,14.</li>
	<li>To assess PGA content and collagen component, observe histologic samples with Sirius red staining by a polarized microscope. PGA remnants are clearly demarcated through birefringence and the remnant area can be quantified based on the cross-sectional area10.</li>
</ol>

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The authors declare they have no competing financial interests.

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 from the perfusion system. The relatively independent perfusion system guarantees the decrease risk of pollution during culture and the placement of OCT guidewire. Meanwhile this intraluminal imaging modality adopted easy and safety monitoring of TEBVs in situs with high resolution approaching that of histopathology, which made the assessment of TEBV growth status more practical and was even expected to be used before or after implant placement.
The current protocol indicates a readily available, fast real-time and nondestructive imaging strategy to evaluate degradable polymer-based engineered vessel development using catheter-based OCT. Through observation of the dynamic process, some main factors affecting vascular engineering, such as contamination or unmatched cell-material interaction led to tissue loss, can be distinguished with early detection. Critical steps to ensure the efficacy of the protocol include the fabrication of NaOH-modified PGA scaffold, successful seeding of HUASMCs in the scaffold, separation sterile culture system from the monitoring system, fast and skilled catheter operation process.
This technique can be utilized to assess degradation states and complex structure of PGA scaffolds blended in with new tissue. The polymeric scaffold with porous network structure degrades gradually and dominates the process of vascular remodeling in the first three weeks, which are important for cell adhesion and extracellular matrix deposition with a three-dimensioned structure for nutrient exchange and as a signal carrier15,16. For quantification of PGA remnants in engineered vessels clearly identified by Sirius red-stained images, the use of polarized microscopes17 in degradable scaffold-based vascular engineering has the potential to become the standard evaluation after cultivation. Hence OCT imaging combined with polarized microscope might serve as qualitative and quantitative methods for assessing PGA degradation in vascular engineering.
A limitation of this technique is the resolution limit to assess cell proliferation, distribution, cell-cell and cell-ECM interaction during engineered vascular remodeling. We hope to find suitable method to investigate TEBVs microstructure at cellular or subcellular level18 and quantify growth kinetics. With quantitative analysis of average optical signals of OCT imaging, we might be more aware of the mechanism of material degradation in vascular engineering. Such experiments are being considered for our future studies.
Overall, our results show that OCT is a readily available, fast real-time and nondestructive imaging strategy to monitor the growth and remodeling of TEBVs. It is utilized to characterize structural architectural features and the long-term remodeling process of engineered vessels. The application of polarized microscope which provided supplementary evidence for the quantification of polymeric remnants in engineered vessels might be useful for assessing scaffold degradation combined with OCT imaging. Taken together, the current protocol holds promising value of OCT for its application in vascular tissue engineering.

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><tbody><tr><td>PGA mesh</td><td>Synthecon</td><td></td><td></td></tr><tr><td>silicone tube</td><td>Cole Parmer</td><td></td><td></td></tr><tr><td>connector</td><td>Cole Parmer</td><td></td><td></td></tr><tr><td>intravascular OCT system</td><td>St. Jude Medical, Inc</td><td>ILUMIEN&amp;trade; OPTIS&amp;trade; SYSTEM</td><td></td></tr><tr><td>scanning electron microscopic</td><td>Philips</td><td>FEI Philips XL-30</td><td></td></tr><tr><td>polarized microscope</td><td>Olympus</td><td>Olympus BX51</td><td></td></tr><tr><td>sutures</td><td>Johnson &amp;amp; Johnson</td><td></td><td></td></tr><tr><td>pulsatile pump</td><td>Guangdong Cardiovascular Institute</td><td></td><td></td></tr><tr><td>LightLab Imaging software</td><td>St. Jude Medical, Inc</td><td></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 during bioreactor cultivation.
Figure 2 showed the process of engineered vascular remodeling through these cross-sectional imaging of tissue microstructure in real time. Geometric morphology was evaluated, including wall thickness, degradable PGA content, and comparison of TEBV thickness in different culture time points as well as presence of pulsatile stimulation. A trend of decreasing thickness and dramatically changes of engineered tissue within the first two weeks of culture was seen, suggesting signal-rich PGA gradual degradation and the structure of new tissue from loose to tight. At 21 days in culture, the vasculature had formed a smooth structure with extracellular matrix evenly distributed and high signal components mostly dissipated. The wall thickness of TEBVs with even signal increased gradually after three weeks of culture. This remodeling occurred earlier and the morphological changes manifested more obviously in the dynamic group (Figure 3). Thereby OCT enables imaging of engineered vascular morphology to be visualized in situ and in real time in the course of long-running culture.
Figure 4 compared OCT images with histopathological finds of TEBV after 4 weeks of culture. Masson&#39;s trichrome staining demonstrates collagen fibers distributed in a certain direction along with PGA remnants in media layer of engineered vessels (Figure 4B). Sirius red staining revealed PGA remnants and collagen component by using a polarized microscope (Figure 4C). Scanning electron micrographs of engineered vessels with compact microstructure were compared with histological assessment (Figure 4D). Taken together, OCT images showed PGA was with different sizes and porous network structure. The structure of PGA scaffold has no obvious change and swollen by direct contact with culture medium in early stage of culture. But signal intensity of PGA was reduced. PGA components was disintegrated and replaced with cells and extracellular matrix. Fewer fragments were seen over four-week period. SEM images of cross-sectional engineered vessels demonstrated fiber rupture to the extension of incubation time. Material and extracellular matrix composites were in honeycomb-like structure with more compact and less transparency.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58040/58040fig1.jpg" /> 
Figure 1. Schematic of tissue engineering vascular culture system, which consisted of a culture chamber in the bioreactor and the perfusion system for OCT imaging. The pulsatile pump provided a stable fluid flow simulating the biomechanical microenvironment. OCT imaging catheter was pulled back in the silicone tube in the culture chamber. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58040/58040fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/58040/58040fig2.jpg" /> 
Figure 2. The microstructure of tissue engineered blood vessels during culture. Over culture time, signal-rich PGA gradually degraded and the structure of new tissue was from loose to tight. TEBVs had a smooth surface and abundant extracellular matrix evenly distributed after four weeks of culture. It showed the process of engineered vascular remodeling through cross-sectional images in real time. This figure has been modified from Chen, W. et al.10 The thickness of silicone tube used here is 0.8mm.&#160;<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58040/58040fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/58040/58040fig3.jpg" /> 
Figure 3. Comparison of TEBV wall thickness change during vascular remodeling in dynamic and static groups obtained from OCT measurements. Error bars indicate standard error. This figure has been modified from Chen, W. et al.10 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58040/58040fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/58040/58040fig4.jpg" /> 
Figure 4. Imaging of biodegradable polymer-based tissue-engineered blood vessels. (A) OCT image of TEBV after four weeks of culture. M: culture medium; S: silicone tube; the thickness of silicone tube used here is 0.8mm. White arrow indicated TEBV. Red arrow indicated PGA fragment. (B) Masson&#39;s trichrome staining demonstrated well-organized collagen fibers along with the residual content of PGA in media layer of engineered vessels. Scale bar=100 &#181;m. (C) Sirius red staining revealed PGA remnants by using a polarized microscope. Green arrow indicates PGA fragment. Scale bar: 100 &#181;m. (D) Scanning electron micrographs of engineered vessel with compact microstructure were showed to compare with histological assessment. Scale bar=50 &#181;m. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/58040/58040fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

5.6K Views.  Guangdong Academy of Medical Sciences, Guangdong General Hospital. Deze methode kan helpen bij het beantwoorden van belangrijke vragen in de vasculaire weefsel-engineering veld, zoals het toezicht op de dynamiek van ingenieur vasculaire groei en remodelleren tijdens de lange termijn cultuur. Het belangrijkste voordeel van het gebruik van optische coherentie tomografie, of OCT, is dat het een direct beschikbare, realtime en niet-destructieve beeldvorming strategie om de structurele kenmerken en het remodelleren proces van engineered schepen te karakteriseren....

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

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological environment, both mechanical and chemical stimulation are required to maintain the proper development and functionality of arterial vessels1,2.
Bioreactor culture systems developed by our group are designed to support vessel regeneration within a precisely controlled chemo-mechanical environment mimicking that of native vessels. Our bioreactor assembly and maintenance procedures are fairly simple and highly repeatable3,4. Smooth muscle cells (SMCs) are seeded onto a tubular polyglycolic acid (PGA) mesh that is threaded over compliant silicone tubing and cultured in the bioreactor with or without pulsatile stimulation for up to 12 weeks. There are four main attributes that distinguish our bioreactor from some predecessors. 1) Unlike other culture systems that simulate only the biochemical surrounding of native blood vessels, our bioreactor also creates a physiological pulsatile environment by applying cyclic radial strain to the vessels in culture. 2) Multiple engineered vessels can be cultured simultaneously under different mechanical conditions within a controlled chemical environment. 3) The bioreactor allows a mono layer of endothelial cells (EC) to be easily coated onto the luminal side of engineered vessels for animal implantation models. 4) Our bioreactor can also culture engineered vessels with different diameter size ranged from 1 mm to 3 mm, saving the effort to tailor each individual bioreactor to fit a specific diameter size. 
The engineered vessels cultured in our bioreactor resemble native blood vessels histologically to some degree. Cells in the vessel walls express mature SMC contractile markers such as smooth muscle myosin heavy chain (SMMHC)3. A substantial amount of collagen is deposited within the extracellular matrix, which is responsible for ultimate mechanical strength of the engineered vessels5. Biochemical analysis also indicates that collagen content of engineered vessels is comparable to that of native arteries6. Importantly, the pulsatile bioreactor has consistently regenerated vessels that exhibit mechanical properties that permit successful implantation experiments in animal models3,7. Additionally, this bioreactor can be further modified to allow real-time assessment and tracking of collagen remodeling over time, non-invasively, using a non-linear optical microscopy (NLOM)8. To conclude, this bioreactor should serve as an excellent platform to study the fundamental mechanisms that regulate the regeneration of functional small-diameter vascular grafts.

Our group has developed a bioreactor culture system that mimics the physiological pulsatile stresses of the cardiovascular system to regenerate implantable small-diameter vascular grafts.

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Doppler Optical Coherence Tomography of Retinal Circulation

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double circular scan (CDCS) that scans around the optic nerve head at 3.40 mm and 3.75 mm diameters. The double concentric circles are performed 6 times consecutively over 2 sec. The CDCS scan is saved with Doppler shift information from which flow can be calculated. The standard clinical protocol calls for 3 CDCS scans made with the OCT beam passing through the superonasal edge of the pupil and 3 CDCS scan through the inferonal pupil. This double-angle protocol ensures that acceptable Doppler angle is obtained on each retinal branch vessel in at least 1 scan. The CDCS scan data, a 3-dimensional volumetric OCT scan of the optic disc scan, and a color photograph of the optic disc are used together to obtain retinal blood flow measurement on an eye. We have developed a blood flow measurement software called "Doppler optical coherence tomography of retinal circulation" (DOCTORC). This semi-automated software is used to measure total retinal blood flow, vessel cross section area, and average blood velocity. The flow of each vessel is calculated from the Doppler shift in the vessel cross-sectional area and the Doppler angle between the vessel and the OCT beam. Total retinal blood flow measurement is summed from the veins around the optic disc. The results obtained at our Doppler OCT reading center showed good reproducibility between graders and methods (&lt;10%). Total retinal blood flow could be useful in the management of glaucoma, other retinal diseases, and retinal diseases. In glaucoma patients, OCT retinal blood flow measurement was highly correlated with visual field loss (R2&gt;0.57 with visual field pattern deviation). Doppler OCT is a new method to perform rapid, noncontact, and repeatable measurement of total retinal blood flow using widely available Fourier-domain OCT instrumentation. This new technology may improve the practicality of making these measurements in clinical studies and routine clinical practice.

Total retinal blood flow is measured by Doppler optical coherence tomography and semi-automated grading software.

Noncontact retinal blood flow measurements are performed with a Fourier domain optical coherence tomography (OCT) system using a circumpapillary double ...

Medicine

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Cancer Research

Automated 3D Optical Coherence Tomography to Elucidate Biofilm Morphogenesis Over Large Spatial Scales

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm morphogenesis, that is the structural diversification of biofilms during community assembly, represents a remarkable challenge across spatial and temporal scales. Here, we present an automated biofilm imaging system based on optical coherence tomography (OCT). OCT is an emerging imaging technique in biofilm research. However, the amount of data that currently can be acquired and processed hampers the statistical inference of large scale patterns in biofilm morphology. The automated OCT imaging system allows covering large spatial and extended temporal scales of biofilm growth. It combines a commercially available OCT system with a robotic positioning platform and a suite of software solutions to control the positioning of the OCT scanning probe, as well as the acquisition and processing of 3D biofilm imaging datasets. This setup allows the in situ and non-invasive automated&#160;monitoring of biofilm development and may be further developed to couple OCT imaging with macrophotography and microsensor profiling.

Microbial biofilms form complex architectures at interphases and develop into highly scale-dependent spatial patterns. Here, we introduce an experimental system (hard- and software) for the automated acquisition of 3D optical coherence tomography (OCT) datasets. This toolset allows the non-invasive and multi-scale characterization of biofilm morphogenesis in space and time.

Biofilms are a most successful microbial lifestyle and prevail in a multitude of environmental and engineered settings. Understanding biofilm ...

Environment

Computed Tomography and Optical Imaging of Osteogenesis-angiogenesis Coupling to Assess Integration of Cranial Bone Autografts and Allografts

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that implantation of a bone autograft would induce greater bone regeneration by abundant blood vessel formation. To investigate the effect of the graft on neovascularization at the defect site, we developed a micro&#8211;computed tomography (&#181;CT) approach to characterize newly forming blood vessels, which involves systemic perfusion of the animal with a polymerizing contrast agent. This method enables detailed vascular analysis of an organ in its entirety. Additionally, blood perfusion was assessed using fluorescence imaging (FLI) of a blood-borne fluorescent agent. Bone formation was quantified by FLI using a hydroxyapatite-targeted probe and &#181;CT analysis. Stem cell recruitment was monitored by bioluminescence imaging (BLI) of transgenic mice that express luciferase under the control of the osteocalcin promoter. Here we describe and demonstrate preparation of the allograft, calvarial defect surgery, &#181;CT scanning protocols for the neovascularization study and bone formation analysis (including the in vivo perfusion of contrast agent), and the protocol for data analysis.
The 3D high-resolution analysis of vasculature demonstrated significantly greater angiogenesis in animals with implanted autografts, especially with respect to arteriole formation. Accordingly, blood perfusion was significantly higher in the autograft group by the 7th day after surgery. We observed superior bone mineralization and measured greater bone formation in animals that received autografts. Autograft implantation induced resident stem cell recruitment to the graft-host bone suture, where the cells differentiated into bone-forming cells between the 7th and 10th postoperative day. This finding means that enhanced bone formation may be attributed to the augmented vascular feeding that characterizes autograft implantation. The methods depicted may serve as an optimal tool to study bone regeneration in terms of tightly bounded bone formation and neovascularization.

Implantation of autologous and allogeneic bone grafts constitute accepted approaches to treat major craniofacial bone loss. Yet the effect of graft composition on the interplay among neovascularization, cell differentiation and bone formation is unclear. We present a multimodal imaging protocol aimed to elucidate the angiogenesis-osteogenesis interdependence at the graft proximity.

A major parameter determining the success of a bone-grafting procedure is vascularization of the area surrounding the graft. We hypothesized that ...

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds during repair, which gives rise to a lack of sufficient blood supply and causes necrosis of the new tissues. Rapid vascularization is a vital prerequisite for new tissue survival and integration with existing host tissue. The de novo generation of vasculature in scaffolds is one of the most important steps in making bone regeneration more efficient, allowing repairing tissue to grow into a scaffold. To tackle this problem, the genetic modification of a biomaterial scaffold is used to accelerate angiogenesis and osteogenesis. However, visualizing and tracking in vivo blood vessel formation in real-time and in three-dimensional (3D) scaffolds or new bone tissue is still an obstacle for bone tissue engineering. Multiphoton microscopy (MPM) is a novel bio-imaging modality that can acquire volumetric data from biological structures in a high-resolution and minimally-invasive manner. The objective of this study was to visualize angiogenesis with multiphoton microscopy in vivo in a genetically modified 3D-PLGA/nHAp scaffold for calvarial critical bone defect repair. PLGA/nHAp scaffolds were functionalized for the sustained delivery of a growth factor pdgf-b gene carrying lentiviral vectors (LV-pdgfb) in order to facilitate angiogenesis and to enhance bone regeneration. In a scaffold-implanted calvarial critical bone defect mouse model, the blood vessel areas (BVAs) in PHp scaffolds were significantly higher than in PH scaffolds. Additionally, the expression of pdgf-b and angiogenesis-related genes, vWF and VEGFR2, increased correspondingly. MicroCT analysis indicated that the new bone formation in the PHp group dramatically improved compared to the other groups. To our knowledge, this is the first time multiphoton microscopy was used in bone tissue-engineering to investigate angiogenesis in a 3D bio-degradable scaffold in vivo and in real-time.

Visualizing Angiogenesis by Multiphoton Microscopy In Vivo in Genetically Modified 3D-PLGA/nHAp Scaffold for Calvarial Critical Bone Defect Repair

Here, we present a protocol to visualize blood vessel formation in vivo and in real-time in 3D scaffolds by multiphoton microscopy. Angiogenesis in genetically modified scaffolds was studied in a murine calvarial critical bone defect model. More new blood vessels were detected in the treatment group than in controls.

The reconstruction of critically sized bone defects remains a serious clinical problem because of poor angiogenesis within tissue-engineered scaffolds ...

Stepwise Cell Seeding on Tessellated Scaffolds to Study Sprouting Blood Vessels

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, structures, phenotypes, and performance depending on each specific perfused tissue. The field of tissue engineering, which aims to repair or replace damaged or missing body tissues, relies on controlled angiogenesis to create a proper vascularization within the engineered tissues. Without a vascular system, thick engineered constructs cannot be sufficiently nourished, which may result in cell death, poor engraftment, and ultimately failure. Thus, understanding and controlling the behavior of engineered blood vessels is an outstanding challenge in the field. This work presents a high-throughput system that allows for the creation of organized and repeatable vessel networks for studying vessel behavior in a 3D scaffold environment. This two-step seeding protocol shows that vessels within the system react to the scaffold topography, presenting distinctive sprouting behaviors depending on the compartment geometry in which the vessels reside. The obtained results and understanding from this high throughput system can be applied in order to inform better 3D bioprinted scaffold construct designs, wherein fabrication of various 3D geometries cannot be rapidly assessed when using 3D printing as the basis for cellularized biological environments. Furthermore, the understanding from this high throughput system may be utilized for the improvement of rapid drug screening, the rapid development of co-cultures models, and the investigation of mechanical stimuli on blood vessel formation to deepen the knowledge of the vascular system.

Engineered tissues heavily rely on proper vascular networks to provide vital nutrients and gases and remove metabolic waste. In this work, a stepwise seeding protocol of endothelial cells and support cells creates highly organized vascular networks in a high-throughput platform for studying developing vessel behavior in a controlled 3D environment.

The cardiovascular system is a key player in human physiology, providing nourishment to most tissues in the body; vessels are present in different sizes, ...

Optical Coherence Tomography Based Biomechanical Fluid-Structure Interaction Analysis of Coronary Atherosclerosis Progression

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis as one of the leading causes of global death, morbidity and economic burden, novel ways of analyzing and predicting its progression are needed. One such computational method is the use of fluid-structure interaction (FSI) to analyze the interaction between the blood flow and artery/plaque domains. Coupled with in vivo imaging, this approach could be tailored to each patient, assisting in differentiating between stable and unstable plaques. We outline the three-dimensional reconstruction process, making use of intravascular Optical Coherence Tomography (OCT) and invasive coronary angiography (ICA). The extraction of boundary conditions for the simulation, including replicating the three-dimensional motion of the artery, is discussed before the setup and analysis is conducted in a commercial finite element solver. The procedure for describing the highly nonlinear hyperelastic properties of the artery wall and the pulsatile blood velocity/pressure is outlined along with setting up the system coupling between the two domains. We demonstrate the procedure by analyzing a non-culprit, mildly stenotic, lipid-rich plaque in a patient following myocardial infarction. Established and emerging markers related to atherosclerotic plaque progression, such as wall shear stress and local normalized helicity, respectively, are discussed and related to the structural response in the artery wall and plaque. Finally, we translate the results to potential clinical relevance, discuss limitations, and outline areas for further development. The method described in this paper shows promise for aiding in the determination of sites at risk of atherosclerotic progression and, hence, could assist in managing the significant death, morbidity, and economic burden of atherosclerosis.

There is a need to determine which atherosclerotic lesions will progress in the coronary vasculature to guide intervention before myocardial infarction occurs. This article outlines the biomechanical modeling of arteries from Optical Coherence Tomography using fluid-structure interaction techniques in a commercial finite element solver to help predict this progression.

In this paper, we present a complete workflow for the biomechanical analysis of atherosclerotic plaque in the coronary vasculature. With atherosclerosis ...

Full-Field Optical Coherence Microscopy for Histology-Like Analysis of Stromal Features in Corneal Grafts

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting factor(s) for success of deep anterior lamellar and penetrating keratoplasty. These are surgical procedures that involve replacing part or all of the diseased corneal layers, respectively, by donated tissue, the graft, taken from a recently deceased individual. However, means to evaluate stromal quality of corneal grafts in eye banks are limited and lack the capability of high-resolution quantitative assessment of disease indicators. Full-field optical coherence microscopy (FF-OCM), permitting high-resolution 3D imaging of fresh or fixed ex vivo biological tissue samples, is a non-invasive technique well suited for donor cornea assessment. Here we describe a method for the qualitative and quantitative analysis of corneal stroma using FF-OCM. The protocol has been successfully applied to normal donor corneas and pathological corneal buttons, and can be used to identify healthy and pathologic features on both the macroscopic and microscopic level, thereby facilitating the detection of stromal disorders that could compromise the outcome of keratoplasty. By improving the graft quality control, this protocol has the potential to result in better selection (and rejection) of donor tissues and hence decreased graft failure.

We describe use of full-field optical coherence microscopy as a method for high quality assessment of corneal donor stroma. This protocol can be used to identify features indicative of health or disease, and is aimed at improving the screening and selection of donor tissues, and hence the results of keratoplasty.

The quality of donor corneal stroma, which makes up about 90% of total corneal thickness, is likely to be one of the main, if not the major, limiting ...

Immunology and Infection

Evaluation of Capillary and Other Vessel Contribution to Macular Perfusion Density Measured with Optical Coherence Tomography Angiography

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries with circulation, and perfusion density, which calculates the percentage of the evaluated area that has circulation. Perfusion density also considers the circulation of vessels larger than capillaries, although the contribution of these vessels to the first is not usually evaluated. As both measurements are automatically generated by optical coherence tomography angiography devices, this paper proposes a method for estimating the contribution of vessels larger than capillaries by using a coefficient of determination between vessel and perfusion densities. This method can reveal a change in the proportion of perfusion density from vessels larger than capillaries, even when mean values do not differ. This change could reflect compensatory arterial vasodilatation as a response to capillary dropout in the initial stages of retinal vascular diseases before clinical retinopathy appears. The proposed method would allow the estimation of the changes in the composition of perfusion density without the need for other devices.

We describe the evaluation of a coefficient of determination between vessel and perfusion density of the parafoveal superficial capillary plexus to identify the contribution of vessels larger than capillaries to perfusion density.

Parafoveal circulation of the superficial retinal capillary plexus is usually measured with vessel density, which determines the length of capillaries ...

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

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