BOTTOM-UP TISSUE ENGINEERING BASED ON MICROSCAFFOLDS PRODUCED BY HIGH-RESOLUTION 3D PRINTING

Jun 29, 2022, 11:50 AM
10m
Room: S3 B

Room: S3 B

Speaker

Kopinski-Grünwald, Oliver (3D Printing and Biofabrication Group, Institute of Materials Science and Technology, TU Wien/ Austrian Cluster for Tissue Regeneration)

Description

Introduction
While the two most-commonly applied approaches in tissue engineering (TE), namely the scaffold-based and the scaffold-free approach come with individual advantages but also drawbacks, Ovsianikov et al. proposed a third strategy for tissue engineering which combines the advantages of both approaches [1].
We propose here to utilize this third strategy to fabricate millimeter-size tissue constructs by fusion of multiple spheroids encaged within microscaffolds. Our approach offers great perspective in regenerating osteo-chondral tissue in vitro, by loading the microscaffolds with human adipose derived stem cells (hASCs) and differentiating them towards osteogenic and chondrogenic phenotypes.
Methodology
The highly porous structures, inspired by the chemical structure of fullerene (C60) referred to as buckyball (BB), were printed using a custom-built Two-Photon polymerization (2PP) system. The 2PP system consisted of a pulsed femtosecond laser operated at 800 nm, that resulted in writing speeds of 600 mm⸱s-1 at an intensity of 85 mW using a 10x objective. The biocompatible and biodegradable, prepolymer called hexa-acrylate-endcapped urethane-based poly-ε-caprolactone (UPCL-6) [2], in combination with 0.5 wt% of photoinitiator M2CMK served as photosensitive resin for 3D-printing. The use of low-adhesive 96-well plates hosting single microscaffolds in each well allowed seeding (4000 cells/well) of hASCs. After 2 days, spheroid formation within the buckyball scaffold was detectable and spheroids were differentiated towards the osteogenic and chondrogenic lineage for 21 days. After differentiation, the microscaffold-reinforced spheroids, referred to as tissue units (TUs) were harvested and merged together in a custom-made cylindrical agarose mold with a diameter of ~1.8 mm and a height of 8 mm for 7 days. 50 TUs differentiated towards the osteogenic lineage were placed at the bottom of the mold, while 50 TUs differentiated towards the chondrogenic lineage were seeded on top of the osteogenic ones to result in an osteochondral interface.
Results and Discussion
The successful differentiation of the spheroids within the microscaffolds towards the osteogenic lineage was verified by calcium deposition quantification, fluorescent calcein green staining, while the successful chondrogenic differentiation was verified by quantification of sulfated glycosaminoglycans and total protein amount. The formation of larger tissue constructs in the range of several millimeters was possible, using these differentiated spheroid-laden BB as “building blocks”.
Conclusion:
Our results indicate that this third TE method could be a promising approach with wide applicability, as the microscaffold can be used for instance to tailor the nature of the final tissue. In addition, the utilization of different cell sources or differentiation into further lineages could pave the way towards a variety of different TE applications.
References:
[1] Ovsianikov, A. et al., Trends in Biotechnology, 2018, doi.org/10.1016/j.tibtech.2018.01.005.
[2] Arslan, A. et al., Mater. Today, 2021, doi: 10.1016/j.mattod.2020.10.005.
Acknowledgements:
This research work was financially supported by the European Research Council (Consolidator Grant 772464 A.O.)

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