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Tissue Engineering

Tissue Engineering - Poster Session E

Poster AA17 - Biofabrication and preliminary in vivo tests of a self-supporting multilayered 3D bioprinted construct for vascular tissue regeneration.

Saturday, October 26, 2024
10:00 AM – 11:00 AM EST
Location: Exhibit Hall E, F & G

Introduction:  Cardiovascular diseases, particularly aortic aneurysms and aortic syndromes, are the leading cause of death globally, claiming around 18 million lives annually. Despite extensive research, therapeutic options remain limited, with treatments since the 1970s primarily relying on synthetic vascular grafts made from materials like PET (Dacron®), PTFE, or ePTFE. These grafts differ mechanically from natural vessels, often causing complications such as pseudo-aneurysms and intimal hyperplasia due to compliance mismatches. The concept of tissue-engineered blood vessels emerged in 1986, leading to innovative techniques such as 3D bioprinting. This method allows for precise, single-step creation of vascular grafts using hydrogels embedded with cells, providing a reproducible platform for bioink application. The study focuses on characterizing a novel developed bioink combining sodium alginate, gelatin, and decellularized extracellular matrix from pig aortas. This bioink aims to replicate the native vascular ECM's hierarchical and chemical structure, promoting cell proliferation and minimizing thrombosis and inflammation risks. The optimized bioink supports prolonged culture in specially designed bioreactors, maintaining the stability and viability of embedded cells.

Materials and

Methods:  Native porcine aorta tissue was decellularized by an optimized protocol to ensure the efficient removal of cellular components while preserving the structural integrity and biochemical properties of the extracellular matrix (ECM). The resulting decellularized ECM (dECM) was cryo-milled into a fine powder under nitrogen to maintain its bioactivity, and then solubilized using pepsin digestion. Different formulations were tested combining different concentrations of gelatin, alginate, solubilized dECM, and cells to determine the optimal composition for replicating the geometrical and structural properties of the native aorta. The constructs were printed using the Inkredible+ bioprinter (Cellink, A Bico Company, Sweden), with different colored bioinks representing the distinct layers of the aortic wall, to highlight different functional areas and to ensure the accurate replication of the native anatomy in the bioprinted constructs. Cell viability and proliferation were evaluated with Live/Dead viability/Cytotoxicity kit (Invitrogen, United States). To assess the biocompatibility and potential immune response of the developed bioink, we conducted in vivo testing using a rat model. This involved subcutaneous implantation of the bioink-based constructs in 6 male, 4-week-old Sprague Dawley (SD) rats, selected to avoid gender-related growth differences. The animal experiment protocol was approved by the Institutional Animal Care and Use Committee at Northwestern University. Post-implantation, the local tissue response was evaluated through biochemical assays and histological analysis of the explanted tissues. This analysis focused on assessing tissue remodeling, cellular infiltration, and the foreign body response to the biomaterials, providing crucial data on the bioink’s performance in a biological setting.

Results, Conclusions, and Discussions: This study developed a novel bioink composed of porcine decellularized extracellular matrix (dECM), gelatin, alginate, and fibroblasts, optimized for 3D bioprinting of multilayered tubular structures (Figure 1). The bioink formulations were evaluated for shape fidelity by analyzing filament diameter, pore index, and filament collapse. A composition of 8% w/v gelatin, 7% w/v alginate, and 2x10^6 fibroblasts provided high geometric fidelity and minimal spreading. Increasing dECM concentration significantly altered the constructs, with 3% w/v dECM selected for creating a 10mm tall tubular segment, maintaining structural accuracy as per the design. The bioink allowed for the successful bioprinting of two distinct layers using different colored bioinks, demonstrating potential for creating separate cell types within the same construct without mixing. In vivo experiments involved subcutaneous implantation in rats, showing an initial acute inflammatory response with increased cell count around the implants. By the second week (Figure 2), collagen organization revealed three distinct regions, indicating ongoing tissue remodeling and integration. The constructs maintained structural fidelity, mechanical properties, and exhibited good cytocompatibility, with high cell viability and proliferation.
The bioink demonstrated mechanical integrity and organized cell layers, essential properties for vascular graft applications. The study also highlighted the bioink's biocompatibility, with minimal adverse immune responses observed in vivo. However, challenges remain, such as fine-tuning the decellularization process to preserve ECM components critical for cell support and avoiding potential damage that could reduce bioink functionality. Future research should explore integrating other cell types, such as smooth muscle cells, to enhance the constructs' functionality.
In summary, this bioink represents a significant advancement in tissue engineering, offering potential for developing complex tissue structures like vascular grafts. Its ability to closely mimic native tissue architecture and functionality marks a promising direction for regenerative medicine, laying the foundation for future clinical applications.

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