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 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.
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.
1. Degradable PGA Scaffold based Tissue-engineered Vessels Culture
2. Performing Optical Imaging with OCT
3. Imaging Analysis
4. Harvest of TEBV and Tissue Processing
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'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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 Please click here to view a larger version of this figure.
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's trichrome staining demonstrated well-organized collagen fibers along with the residual content of PGA in media layer of engineered vessels. Scale bar=100 µm. (C) Sirius red staining revealed PGA remnants by using a polarized microscope. Green arrow indicates PGA fragment. Scale bar: 100 µm. (D) Scanning electron micrographs of engineered vessel with compact microstructure were showed to compare with histological assessment. Scale bar=50 µm. Please click here to view a larger version of this figure.
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.
The authors have nothing to disclose.
We would like to acknowledge the Science and Technology Planning Project of the Guangdong Province of China (2016B070701007) for supporting this work.
PGA mesh | Synthecon | ||
silicone tube | Cole Parmer | ||
connector | Cole Parmer | ||
intravascular OCT system | St. Jude Medical, Inc | ILUMIEN™ OPTIS™ SYSTEM | |
scanning electron microscopic | Philips | FEI Philips XL-30 | |
polarized microscope | Olympus | Olympus BX51 | |
sutures | Johnson & Johnson | ||
pulsatile pump | Guangdong Cardiovascular Institute | ||
LightLab Imaging software | St. Jude Medical, Inc |