生体活性人工血管の開発と血管組織修復のための免疫調節の役割の解明

2021 Fiscal Year Annual Research Report

• Project/Area Number: 21F21353
• Allocation Type: Single-year Grants
• Research Institution: Kyushu University
• Principal Investigator: 井嶋 博之 九州大学, 工学研究院, 教授 (10274515)
• Co-Investigator(Kenkyū-buntansha): SHAFIQ MUHAMMAD 九州大学, 工学(系)研究科(研究院), 外国人特別研究員
• Project Period (FY): 2021-11-18 – 2024-03-31
• Keywords: Hybrid vascular graft / Decellularized ECM / Polycaprolactone / Nanofibrous Membrane / Tubular scaffold / Electrospun membrane

Outline of Annual Research Achievements

Cardiovascular diseases (CVD) account for the huge morbidity and mortality worldwide, which necessitate vascular grafting. While autografts are gold-standard treatment regimes, their use is hampered owning to their limited availability and immunogenicity complications. Alternatively, synthetic vascular grafts based on polycaprolactone (PCL) and poly(L-lactide-co-ε-caprolactone) (PLCL) have been widely studied, however, the lack of bioactive cues and poor blood compatibility limit their applicability. Hybrid vascular grafts based on decellularized extracellular matrix (dECM) and synthetic polymers may be a viable option to address these limitations, which can be further functionalized with endothelium-mimicking biological molecules, including heparin and growth factors to further induce tissue repair. Consequently, the overarching goal of this research is to produce hybrid electrospun scaffolds consisting of polycaprolactone/gelatin (PCL/Gel) and decellularized extracellular matrix (dECM). To meet these goals, we have attempted the fabrication of core/shell type polycaprolactone/gelatin based electrospun nanofibers and tubular-shaped scaffolds and evaluated their morphology. Additionally, the decellularization of different types of tissues, such as porcine liver and brain was performed. To further mimic the native vasculature, a systematic survey was performed to choose an optimum method for the decellularization of porcine aorta. The preparation for several experiments, such as in vitro cell culture, histological analysis, and in vitro biochemical assays was performed.

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

As the objective of this research is to develop hybrid electrospun scaffolds for vascular reconstruction, a systematic survey was performed to screen the potential candidates for electrospinning and methodologies for the decellularization of different types of tissues, including porcine skin and brain. Based on such preliminary survey, it was found that so far only a limited research has been performed on the fabrication of core/shell type electrospun scaffolds, particularly only few studies have been performed on the hybrid scaffolds comprising of PCL/Gel and dECM for vascular reconstruction. As preliminary experiments, core/shell type PCL/Gel scaffolds were successfully prepared, which displayed nanofibrous and porous morphology. Besides, the decellularization of porcine heart and brain was performed, which revealed successful decellularization along with the preserved extracellular matrix (ECM). Hybrid scaffolds based on PCL/Gel and dECM were also successfully prepared. As we plan to conjugate endothelium-mimicking molecules with scaffolds, heparin was successfully immobilized and evaluated by toluidine blue (TB) assay. Conclusively, in this fiscal year, basic studies related to the fabrication of electrospun scaffolds and decellularization was successfully performed, which would lay a sound framework for the ongoing studies to satisfactorily accomplish the objectives in a stipulated time frame.

Strategy for Future Research Activity

Decellularization of tissues: Different types of tissues, such as skin, aorta, and heart will be decellularized and characterized by using a series of experiments, such as hematoxylin and eosin (H&E) staining as well as GAG, deoxyribonucleic acid (DNA), and extracellular matrix (ECM) content. Briefly, the tissues will be acquired from the slaughterhouse and subjected to decellularization. The resulting dECM will then be converted into powder and stored for subsequent blending with the polymer matrix for electrospinning.
Fabrication of Nanofibrous Membranes and tubular scaffolds: Electrospun membranes and tubular-shaped scaffolds will be prepared. Briefly, core/shell type scaffolds consisting of PCL/Gel based core and Gel/dECM based shells hybrids will be fabricated. The morphology of membranes will be assessed by using SEM. Next, heparin will be conjugated with the membranes and growth factors, such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) will be electrostatically immobilized and analyzed. The bioactivity of membranes will be assessed in vitro by using different types of assays, including live/dead assay, cell proliferation assay, and so on.
Assessment of the Biocompatibility of Scaffolds In Vivo: The scaffolds will be further evaluated in vivo. A series of histological and immunohistochemical assays will be performed to evaluate cellular infiltration, inflammatory response, and tissue regeneration. The quantitative analysis of the obtained results will further be carried out.