gms | German Medical Science

Objectives: Bone tissue engineering approaches usually include isolation and in vitro propagation of autologous osteoblasts or their precursor cells, followed by seeding on different resorbable scaffolds. The aim of the present study was to investigate, if differentiation of the complete tissue fragments with inhomogeneous cell populations could be practically realizable.

Methods: In this study, 3D collagen-I culture of human fat tissue fragments was established according to Sonoda et al., 2008 and we found an increasing cell viability over 4 weeks, detected by WST-1 assay. The cultures were cultivated either in growth medium or in osteogenic medium (OM) with or without addition of Bone Morphogenetic Proteins (BMPs): BMP-2, BMP-7 or BMP-9. Evaluation of tissue morphology and Ca²⁺ deposition was performed by haematoxylin and eosin, Alizarin red S and von Kossa staining of histological slices. Expression of osteoblast specific proteins was performed after 1, 2 and 4 weeks of culture at the mRNA level by the quantitative RT-PCR, and at the protein level by the immunofluorescence.

Results and Conclusion: Under osteogenic conditions, the Ca²⁺ depositions were observed after 2 weeks of incubation and clearly increased by addition of each type of BMPs, reaching a maximum at week 4. mRNA levels of alkaline phosphatase (APL) and osteocalcin (OC) slightly increased under osteogenic conditions (OM). Addition of BMP-2, BMP-7 or BMP-9 caused significantly higher expression levels of ALP and OC. BMP-9 seems to be the most effective osteogenic inducer in this system.

Furthermore, it was shown by immunofluorescence that all the cultures were positive for OC and osteopontin (OP) proteins, whereas only differentiated tissues were positive for sclerostin, the marker of osteocytes. Addition of BMPs increased expression of OC, OP and sclerostin significantly.

The tested 3D collagen-I fat culture system is a useful tool to study differentiation of 3D tissue constructs. Human fat tissue fragments may be considered as an alternative to stem cell isolation approaches in bone tissue engineering.