Optimization of perfusion system for bone tissue growth on peptide hydrogel

The goal of our research is to develop a new scaffold for bone tissue healing which is biocompatible, biodegradable, non-immunogenic and supports acceptance, proliferation and differentiation of cells. The aim of this study was to establish 3D culture of mesenchymal stem cells (MSC) in a perfusion bioreactor using peptide hydrogel RADA16. Hydrogel RADA16 is a new class of scaffolds made of 1% peptide and 99% water. In the presence of salt it polymerizes and forms 3D network which mimics extracellular matrix. However, even after polymerisation, hydrogel is too soft to resist perfusion. To increase the viscosity of scaffold we used 1% hydrogel RADA and we modified perfusion system to ensure proper support for hydrogel. We defined the optimal perfusion rate of 0.1ml/min. Of total number of cells inserted into the bioreactor, 95% of the cells attached to scaffold. It was also found that hydrogel does not retain minerals within the scaffold, but encourages their production. Due to perfusion, minerals precipitate on the bottom of the bioreactor. Bone graft is analyzed after 21 days by histology and qPCR. Osteoblast differentiation was comfirmed by gene expression of bone markers BSP and DMP1. Hematoxylin/eosin staining confirmed the presence of cells on outer layer of bone graft and connective tissue in the inner part. Therefore, we conclude that RADA16 can be used in perfusion bioractor for the bone tissue formation, with some modification of the system and with lower perfusion rate. The remaining problem is still the low retention of minerals within the graft.