Abstract
Introduction Tissue engineering aims to produce a cellular structure in an extracellular matrix, which when implanted heals tissue defects.
To tissue-engineer bone suitable cells need to be grown on a scaffold. In this study we grew human marrow cells as they can differentiate into osteoblasts, on porous hydroxyapatite (HA) scaffolds, as this is osteoconductive, allows cell penetration and in growth of capillaries after implantation.
Increased extravascular perfusion through bone increases new bone formation. So we reproduced these physiological conditions in our novel bioreactor by perfusing scaffolds at 6ml/hr.
Hypotheses 1. Culture in our bioreactor improved cell penetration through HA scaffolds compared to static conditions. 2. Human mesenchymal stem cells (MSCs) cultured in our bioreactor differentiated into osteo-blasts and produced bone extracellular matrix.
Method MSCs were isolated from 8 human bone marrow aspirates taken from patients following informed consent. For each experiment 16 scaffolds were seeded with MSCs and comparisons were made between the two conditions. After 7 days culture the scaffolds were sectioned longitudinally and the number of cells at increasing depths were counted. The scaffolds were observed under SEM & TEM. Osteoblastic markers ALP and type I pro-collagen (PICP) were measured.
Results Penetration of cells through the scaffolds was significantly greater when cultured in the bioreactor.
After 14 days in bioreactor culture the HA was covered with cuboidal cells, consistent with osteoblasts, however in static culture cells remained fibroblastic. TEM results showed that MSCs in the bioreactor produced organised collagen matrix after 21 days and osteoid by 28 days, but no collagen matrix was observed following static culture.
ALP and PICP were significantly greater over 15 days culture when in our bioreactor.
Conclusions These results show that when MSCs were cultured in our bioreactor they attached and penetrated through porous HA scaffolds, whereas in static conditions few cells penetrated below 2mm. Our bioreactor significantly improved 3-dimensional growth, resembling tissue.
Moreover, MSCs grown on HA in the bioreactor produced significantly more ALP and PICP indicating osteoblastic differentiation. Furthermore, bone osteoid was produced.
Therefore this culture method could be use to convert autologous MSCs from human marrow into tissue-engineered bone which could be used to heal defects after tumor excision.
Theses abstracts were prepared by Professor Roger Lemaire. Correspondence should be addressed to EFORT Central Office, Freihofstrasse 22, CH-8700 Küsnacht, Switzerland.
