# Design and Validation of a Multi-Modal Bioreactor System: Assessing the Effects of Perfusion and Cyclic Tensile Stimulation on Mechanical and Biological Properties of 3D-Printed Missing-Rib Auxetic Scaffolds

## TL;DR

A new bioreactor system was developed and tested to improve the mechanical and biological performance of 3D-printed auxetic scaffolds used in tissue engineering.

## Contribution

The study introduces a custom multi-modal bioreactor that combines perfusion and cyclic tensile stimulation for tissue scaffold maturation.

## Key findings

- The bioreactor did not reduce the scaffold's yield strength or elastic modulus but decreased its ultimate tensile strength by 22.5%.
- Fibroblast cellular responses, including DNA and collagen production, were significantly improved with bioreactor use.
- The system validates the potential of multi-modal stimulation to enhance scaffold biofunctionality for tissue-specific applications.

## Abstract

Bioreactors used for the maturation of cell-seeded tissue-engineered scaffolds should essentially mimic the dynamic in vivo environments experienced by the native tissues they intend to substitute. In addition to perfusion of growth medium to facilitate continuous mass transfer, application of appropriate mechanical stimulation is important to enhance cellular responses in scaffolds for tissues such as tendons, skin, and cardiac muscle that experience dynamic loading. This study focuses on the development of a multi-modal custom bioreactor capable of applying cyclic tensile stimulation and perfusion within physiologically relevant ranges while minimizing shear stress detrimental to cells seeded on scaffolds. To validate the bioreactor design and operation, we assessed the effects of tensile stimulation (0.1 Hz, 2000 cycles/day) and perfusion (media flow rate = 0.15 mL/min) over 21 days on the biofunctional performance of 3D-bioplotted polycaprolactone (PCL) auxetic scaffolds with a representative design (missing-rib pattern) characterized by negative Poisson’s ratio similar to the aforementioned soft tissues. The scaffold had a tensile yield strain of 9.14%, yield strength of 0.25 MPa, elastic modulus of 2.85 MPa, and ultimate tensile strength (UTS) of 1.32 MPa. The application of perfusion and tensile stimulation (0–5% cyclic strain) for 21 days did not adversely affect the yield strength and elastic modulus of the scaffold but affected its UTS (22.5% decrease) compared to the control cultured without perfusion or stimulation. Notably, it resulted in significantly improved fibroblast cellular responses (DNA = 29 µg/g sample and collagen = 371.78 µg/g sample) compared to the control (7.52 µg/g sample and 163.51 µg/g sample, respectively). These results validate the bioreactor system operation and the ability of multi-modal stimulation to control biofunctional responses of auxetic scaffolds, which will serve as the basis for future studies that will optimize auxetic scaffold design and dynamic culture parameters for NPR tissue-specific applications.