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Method Article
Here, a protocol is presented for the implantation of a tissue-engineered vascular graft into the mouse carotid artery using the cuff technique, providing a suitable animal model for investigating vascular tissue regeneration mechanisms.
The development of small-diameter vascular grafts has been a global endeavor, with numerous research groups contributing to this field. Animal experimentation plays a pivotal role in assessing the efficacy and safety of vascular grafts, particularly in the absence of clinical applications. Compared to alternative animal models, the mouse implantation model offers several advantages, including a well-defined genetic background, a mature method for disease model construction, and a straightforward surgical procedure. Based on these advantages, the present study devised a simple cuff technique for the implantation of tissue-engineered vascular grafts in the mouse carotid artery. This technique began with the fabrication of polycaprolactone (PCL) small-diameter vascular grafts via electrostatic spinning, followed by the seeding of macrophages onto the grafts through perfusion adsorption. Subsequently, the cellularized tissue-engineered vascular grafts were transplanted into the mouse carotid artery using the cuff technique to evaluate patency and regenerative capability. After 30 days of in vivo implantation, vascular patency was found to be satisfactory, with evidence of neo-tissue regeneration and the formation of an endothelial layer within the lumen of the grafts. All data were analyzed using statistical and graphing software. This study successfully established a mouse carotid artery implantation model that can be used to explore the cellular sources of vascular regeneration and the mechanisms of action of active substances. Furthermore, it provides theoretical support for the development of novel small-diameter vascular grafts.
The prevalence and mortality of cardiovascular diseases are increasing globally, representing a significant public health concern1. Vascular bypass grafting is an effective intervention for severe coronary heart disease and peripheral vascular disease2. The use of artificial vascular grafts with diameters exceeding 6 mm has been well-documented in clinical settings. Conversely, those with a diameter below 6 mm are prone to thrombosis and intimal hyperplasia, which can lead to a considerable risk of restenosis3. Despite significant advancements in the research and development of small-diameter vascular grafts in recent years, with several products approaching clinical application, multiple challenges remain4,5. These include a relatively low long-term patency rate, limited vascular regeneration, and an insufficient understanding of the regeneration mechanism.
Preclinical evaluation of novel small-diameter vascular grafts relies on in vivo implantation in various animal models. The most commonly used models include the sheep carotid artery, dog femoral artery, rabbit carotid artery, and rat abdominal artery implantation models6,7,8,9. The patency of vascular grafts can be assessed in medium- to large-sized animals, such as sheep, pigs, and dogs. However, these studies involve substantial costs due to the expertise and equipment required. Additionally, their technical complexity poses a challenge to implementation. In contrast, small animal models such as rabbits and rats lack well-established transgenic species with clearly defined genetic backgrounds, presenting a significant obstacle in studying vascular regeneration mechanisms.
Compared to the aforementioned animal models, the mouse model offers a relatively straightforward surgical procedure, a well-established methodology for generating genetically engineered mice, and a clearly defined genetic background. However, the small diameter of mouse blood vessels makes end-to-end anastomosis in vascular grafting technically complex, requiring significant expertise and yielding a relatively low success rate. To reduce the complexity of the procedure and improve the success rate of vascular graft implantation, the present study employed the cuff technique in a mouse carotid artery implantation model.
Following in vivo implantation, vascular grafts can recruit endogenous cells that contribute to vascular tissue regeneration. The presence of these cells facilitates the endothelialization and regeneration of the smooth muscle layer of grafts.10. However, the source and type of cells involved in vascular tissue regeneration remain unclear, and multiple competing theories are under investigation11. Among these, research has focused on the roles of inflammatory and stem cells. Breuer et al. seeded human bone marrow-derived monocytes (hBMCs) onto vascular grafts and found that the seeded cells recruited host cells into the graft wall through the release of monocyte chemoattractant protein-1 (MCP-1), thereby promoting vascular tissue regeneration12. In this study, an efficient perfusion adsorption cell-seeding method was proposed and successfully used to seed macrophages onto polycaprolactone (PCL) small-diameter vascular grafts. Following implantation, these cells exhibited sustained viability.
This article details the methodology for preparing tissue-engineered vascular grafts and the carotid artery implantation procedure in mice using the cuff technique. The process begins with the fabrication of PCL small-diameter vascular grafts with defined parameters via electrostatic spinning. Subsequently, grafts deemed suitable for implantation undergo mechanical testing. Macrophages are then seeded onto the vascular grafts using the perfusion adsorption method. Finally, macrophage-seeded vascular grafts are implanted into the mouse carotid artery using the cuff technique, and the patency and regenerative properties are analyzed after one month of in vivo implantation.
This technique has the potential to enhance the efficacy and success rates of vascular grafting in mouse models. Furthermore, the model can be used to investigate the mechanisms underlying cell sources, pivotal genes, and active factors in vascular regeneration, providing theoretical and methodological support for the functional modification and development of novel small-diameter vascular grafts.
All animal procedures were approved by the Animal Experiments Ethical Committee of the Institute of Radiation Medicine, Chinese Academy of Medical Sciences, and complied with the Guidelines for the Care and Use of Laboratory Animals. Male C57BL/6 mice, 6-8 weeks old, with a body weight of 25-30 g, were used in this study. Details of the reagents and equipments used in this study are listed in the Table of Materials.
1. Fabrication of small-diameter vascular grafts
NOTE: Fabricate small-diameter PCL vascular grafts using the electrospinning technique13.
2. Seeding of macrophages onto vascular grafts
NOTE: Ensure that all solutions and materials are sterile. Conduct all operations within the cell culture room.
3. Mouse carotid artery implantation model
NOTE: Maintain a sterile surgical area for animal procedures. Sterilize all surgical instruments and disposables prior to surgery.
4. Post-procedural care and analysis
Small-diameter vascular grafts with different parameters were successfully prepared via electrospinning. SEM images revealed that the fibers were uniformly distributed and exhibited an irregular arrangement within the graft wall, with the presence of pore structures (Figure 4). As the concentration of PCL increased, both the fiber diameter and pore size increased. Specific values for each vascular graft group are presented in Table 2. The results of mechan...
The use of the cuff technique for implanting tissue-engineered vascular grafts in the mouse carotid artery represents a significant advancement in cardiovascular research15. The critical steps of this technique include cell seeding and graft implantation. This study employed a perfusion adsorption approach to enhance macrophage seeding density to address issues related to non-uniform cell seeding and low cell viability. This method allowed macrophages to infiltrate the vascular graft wall and dist...
The authors have no conflicting financial interests.
Funding for this study was provided by the National Natural Science Foundation of China projects (no. 32101098, 32071356, and 82272158) and the CAMS Innovation Fund for Medical Sciences (no. 2022-I2M-1-023).
Name | Company | Catalog Number | Comments |
1% penicillin-streptomycin | Solarbio | P1400 | |
10% fetal bovine serum | Gibco | A5256701 | |
4% paraformaldehyde | Solarbio | P1110 | |
4',6-Diamidino-2-Phenylindole (DAPI) | SouthernBiotech | 0100-20 | |
Alcohol | Tianjin Chemical Reaggent Company | 1083 | |
Anti-Mouse CD31 primary antibody | BD Bioscience | 553370 | |
Arterial clips | RWD Life Science | R31005-06 | |
C57BL/6 mice | Beijing Vital River Laboratory Animal Technology Company | ||
Dulbecco's modified eagle medium (DMEM) | Gibco | 11966025 | |
Electrostatic spinning machine | Yunfan Technology | DP30 | |
Goat anti-rat IgG (Alexa Fluor 555) | Invitrogen | A-21434 | |
Hematoxylin and eosin (H&E) | Solarbio | G1120 | |
Hexafluoroisopropanol (HFIP) | McClean | H811026 | |
Iodophor | LIRCON | V273068 | |
Microscissors | World Precision Instruments | 14124 | |
Microtweezers | World Precision Instruments | 500338 | |
Normal goat serum | Boster | AR0009 | |
Normal saline | Cisen Pharmaceutical company | H20113369 | |
Nylon tube for cuff | Portex | ||
Optimal cutting temperature compound (OCT) | Sakara | 4583 | |
Pentobarbital sodium | Sigma | P3761 | |
Phosphate Buffered Saline (PBS) | Solarbio | P1003 | |
Poly(ε-caprolactone) (PCL) pellets (Mn = 80,000) | Sigma | 704067 | |
RAW264.7 macrophages | Biyuntian Biotechnology | ||
Scanning electron microscope (SEM) | Zeiss | PHENOM-XL-G2 | |
Surgical sutures 6-0 | Ningbo Chenghe microapparatus factory | 220919 | |
Surgical sutures 9-0 | Ningbo Chenghe microapparatus factory | 221006 | |
Syringe | Changqiang Medical Devices | 0197 | |
Tensile testing machine | Instron | WDW-5D |
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