Investigation of structural and mechanical properties of bioprinted

cell/hydrogel constructs for fabrication of biohybrid heart valves

Shannon Anna Jung, Institue for Technical and Macromolecular Chemistry, Research Area Functional and Interactive

Polymers, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany & DWI - Leibniz Institute for Interac-

tive Materials, RWTH Aachen University, Forckenbeckstraße 50, 52074 Aachen, Germany, sjung@dwi.rwth-aachen.de

Shannon Anna Jung1,2, Miriam Aischa Al Enezy-Ulbrich1,2, Svenja Wein3,4, Christian Böhm5, Norina Labude-Weber3,4,

Hanna Malyaran3,4, Stefan Jockenhoevel5,6, Sabine Neuss3,4, Andrij Pich1,2

1 Institute for Technical and Macromolecular Chemistry, Research Area Functional and Interactive

Polymers, RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany 2 DWI - Leibniz Institute for Interactive Materials, RWTH Aachen University,

Forckenbeckstraße 50, 52074 Aachen, Germany 3 Helmholtz Institute for Biomedical Engineering, BioInterface Group, RWTH Aachen University,

Pauwelsstrasse 20, 52074 Aachen, Germany 4 Institute of Pathology, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany 5 AME BioTex – Biohybrid & Medical Textiles, RWTH Aachen University, Forckenbeckstraße 55,

52070 Aachen, Germany 6 Aachen-Maastricht Institute for Biobased, Sciences, Faculty of Science and Engineering, Maastricht,

Netherlands

Introduction

As a result of global rise in ageing populations, heart valves diseases have become a majorhealth issue world-wide. The

most widely used treatment for heart valve diseases are surgical replacement strategies with mechanical or biological

heart valves implants. However, existing heart valve implants exhibit several drawbacks including the formation of blood

clots (mechanical implants), poor durability and limited life-time, absence of repair and remodel ability. In the current

work, we focus on a tissue engineered heart valve supporting both biocompatibility and reproducibility of implants.

Methods

Here, three-dimensional cell/hydrogel constructs are synthesized for the application in biohybrid heart valves. Fibrin-

based hydrogels with L929 cells are used for bioprinting process to obtain cell/hydrogel constructs and their mechanical

and structural properties are systematically investigated. The hydrogels are synthesized from natural fibrin and synthetic

linear reactive copolymers based on poly(N-vinylpyrrolidone) or fibrin-binding peptide and can provide a three-dimen-

sional environment for cell proliferation and growth. As standard cell line for cytotoxicity assays related to the ISO 10993-

5, L929 cells are used for initial experiments to determine the influence of the cells on the mechanical properties of the

hydrogels and for the optimization of the bioprinting process.

Results

Rheological experiments prove that the cell concentration has no significant influence on the hydrogels mechanical prop-

erties. Moreover, analysis of the cell/hydrogel constructs with confocal, 2-photon and scanning electron microscopy show

a homogenous distribution of cells inside the hydrogel. In addition, viability assays of cells confirm the suitability of the

fibrin-based hydrogel as a scaffold for embedding cells.

Furthermore, the possibilities to fabricate cell/hydrogel constructs in different geometries (e.g. cylinder or square) are

explored using an extrusion based bioprinter. To enhance the printability of fibrin-based hydrogels, a support bath based

on freeform reversible embedding of suspended hydrogel (FRESH) printing technique is used. The cell viability of the

bioprinted construct was analyzed with live/dead staining and 2-photon microscopy and demonstrated cytocompatibility.

Conclusion

Bioprinted fibrin hydrogel-cell constructs exhibit tunable mechanical properties, controlled porosity, excellent cell-com-

patibility and are promising materials for the fabrication of biohybrid heart valves.

S177Abstracts – BMT 2021 – Hannover, 5 – 7 October • DOI 10.1515/bmt-2021-6024 Biomed. Eng.-Biomed. Tech. 2021; 66(s1): S177–S178 • © by Walter de Gruyter • Berlin • Boston

Special Session DFG PAK 961 followed by P1:

Functional fibrin-based hydrogels for controlling cell/biomaterial

interactions in biohybrid implants

Svenja Wein1,2, Carina Schemmer1,2, Hanna Malyaran1,2, Norina Labude-Weber1,2, Stephan Rütten3, Nicole Terefenko4,5,

Miriam Al Enezy-Ulbrich4,5, Andrij Pich4,5, Sabine Neuss1,2

1 Helmholtz Institute for Biomedical Engineering, BioInterface Group, RWTH Aachen University,

Pauwelsstrasse 20, 52074 Aachen, Germany 2 Institute of Pathology, RWTH Aachen University, Pauwelsstrasse 30, 52074 Aachen, Germany 3 Electron Microscopic Facility, University Clinics, RWTH Aachen University, Pauwelsstrasse 30, 52074

Aachen, Germany 4 DWI - Leibniz Institute for Interactive Materials, RWTH Aachen University, Forckenbeckstraße 50,

52074 Aachen 5 Institute for Technical and Macromolecular Chemistry, Research Area Functional and Interactive

Polymers, RWTH Aachen University, Forckenbeckstraße 50, 52074 Aachen

Introduction

Today's implant research faces the challenge of improving both the biocompatibility and reproducibility of implants.

Biohybrid implants consisting of a technical component and a patient-specific component are suitable for this purpose.

The aim of the consortium is the development of a biohybrid heart valve based on a fibrin-gel matrix with textile rein-

forcement. For this purpose, a functional toolbox for fibrin-based hydrogels is developed in our subproject and the direct

cell/biomaterial interaction and long-term behavior of the cells in the in vitro implant is investigated.

Methods (14 pt bold)

More concrete new biohybrid hydrogels from fibrinogen and poly(N-vinylcaprolactam) copolymers are used. For later

application of the heart valve, smooth muscle cells are required. Therefore, human stem cells are cultured inside the

hydrogels for myogenic differentiation induced by the addition of the specific growth factors TGF-β1 and BMP4 over 21

days.

We focus on the contractile phenotype of smooth muscle cells as it is mandatory for functional heart valves.

Vascularization is essential for the supply of the tissue engineering construct after. Thus, the formation of capillary-like

structures by endothelial cells in contact with feeder layers of stem cells within the gel is analyzed.

The analysis is performed by RT-qPCR, immunofluorescence and SEM as well as two-photon microscopy, with emphasis

on the expression of contractile and synthetic markers. For vascularization the marker CD31 is analyzed.

Results (14 pt bold)

Initial experiments show long-term stable hydrogels, which support the proliferation of human stem cells, so that the

myogenic differentiation of the cells in the gels can be achieved in a subsequent step. The expression of contractile mark-

ers is demonstrated after 21 days. Successful angiogenesis was carried out on TCPS and gel. Particularly noteworthy here

is the parallel alignment of the capillaries, which can only be observed on the gel. It can be assumed that the gel applies

a mechanical traction to the cells and thus brings about the alignment. Further analyses are currently being performed.

Conclusion (14 pt bold)

At this stage, our research shows that compared to conventional heart valves, biohybrid heart valves offer the decisive

advantage of personalization using the patient's own stem cells and thus represent a future-oriented technology. The tissue

engineered heart valve is to be specially tailored to the patient's cell type, so that in future it is possible to reduce the

body's rejection reactions in this way.

S178Abstracts – BMT 2021 – Hannover, 5 – 7 October • DOI 10.1515/bmt-2021-6024 Biomed. Eng.-Biomed. Tech. 2021; 66(s1): S177–S178 • © by Walter de Gruyter • Berlin • Boston