An optimized 3D-printed perfusion bioreactor for homogeneous cell seeding in bone substitute scaffolds for future chairside applications

Nadja Engel,Bernhard Frerich,Carsten Fechner,Bernd Joachim Krause,Klaus-Peter Schmitz,Jan Liese,Carina Bergner,Annika Voges,Robert Ott,Stefan Siewert,Jan Stenzel,Valeria Khaimov

doi:10.1038/s41598-021-01516-8

Abstract

A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailor-made bioreactor design. A perfusion system for efficient, homogeneous, and rapid seeding with human adipogenic stem cells in bone substitute scaffolds was designed. Variants concerning medium inlet and outlet port geometry, i.e. cylindrical or conical diffuser, cell concentration, perfusion mode and perfusion rates were simulated in silico. Cell distribution during perfusion was monitored by dynamic [18F]FDG micro-PET/CT and validated by laser scanning microscopy with three-dimensional image reconstruction. By iterative feedback of the in silico and in vitro experiments, the homogeneity of cell distribution throughout the scaffold was optimized with adjustment of flow rates, cell density and perfusion properties. Finally, a bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode. During this short period of time, the cells initially adhere within the entire scaffold and stay viable. After two weeks, the formation of several cell layers was observed, which was associated with an osteogenic differentiation process. This newly designed bioreactor may be considered as a prototype for chairside application.

Highlights

A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailormade bioreactor design
In order to allow for a homogenous stem cell distribution, the geometry of the bioreactor was optimized so that a laminar, distributed volume flow throughout the bioreactor-scaffold system was achieved
The numerical simulations revealed that the connector geometry at the inlet and outlet of the bioreactor had a decisive influence on the homogeneity of the flow within the scaffold

Summary

Introduction

A clinical implementation of cell-based bone regeneration in combination with scaffold materials requires the development of efficient, controlled and reproducible seeding procedures and a tailormade bioreactor design. A bioreactor with a conical diffusor geometry was developed, that allows a homogeneous cell seeding (hoover coefficient: 0.24) in less than 60 min with an oscillating perfusion mode During this short period of time, the cells initially adhere within the entire scaffold and stay viable. A successful bone formation takes place over a maximum gap distance of 2–3 mm within the bone, vertical and voluminous augmentations are associated with higher complication rates since bone has a limited capacity for spontaneous healing of critical defects caused by injury, inflammation or therapeutic resection[5,6] It has been proven in animal and clinical studies that the combination of osteogenic progenitor cells and bone graft substitutes can significantly increase their efficiency and regenerative v alue[7,8]. In order to save experiments, virtual computational modeling was used beforehand, and the results of the modeling were iteratively compared with the experiment

Methods

Results

Discussion

Conclusion