gms | German Medical Science

Objectives: Bioceramic scaffolds consisting of hydroxylapatite and beta tricalcium phosphate were developed through the rapid prototyping technique at the Friedrich-Baur BioMed Center. In the present study it was tested in vitro if the new scaffolds support viability of the human adipose-derived stem cells and demonstrate the bone inducing properties.

Methods: Adipose-derived stem cells were isolated from human subcutaneous fat tissue and tested for the CDs 34, 39, 44, 45, 73, 90, 105, 166 as well as for the endogenous expression of octeocalcin (OC), osteopontin (OP), dentin matrix protein 1 (DMP-1) and sclerostin in vitro by means of immunofluorescence. Then, the cells were seeded on the bioceramic scaffolds and incubated statically in cell growth medium (GM), osteogenic medium (OM) or OM with addition of Bone Morphogenetic Protein-2 (OM+BMP-2) over 4 weeks. Cell viability and proliferation were examined by combination of WST-1 test and Pico Green DNA-Assay. Expression of osteoblast specific proteins was evaluated after 1, 2 and 4 weeks of culture at the mRNA level (alkaline phosphatase (ALP), OC) by the quantitative RT-PCR as well as at the protein level (OC, OP, DMP-1, sclerostin) by means of immunofluorescence. Histological slices of the decalcified constructs stained with haematoxylin and eosin were examined for the cell morphology and distribution in the scaffold.

Results and Conclusion: In monolayer, the adipose-derived stem cells were positive for CD 44, 73, 90, 105, 166; slightly and partly positive for CD 34 and 39; and negative for CD 45. Furthermore, the cells were positive for osteoblast specific proteins OC, OP, DMP-1, and negative for sclerostin. Seeded on the scaffolds, the cells showed increasing viability and proliferation rates over 4 weeks. mRNA levels of ALP increased significantly in OM and OM+BMP-2 groups during the whole period of incubation; in the GM group at week 4 only. Expression of OC was significantly higher in the OM+BMP-2 group during the whole period of observation. Histology demonstrated the homogeneous distribution of the cells in the scaffolds and correlated with their proliferation rates. Immunofluorescence signals of intracellular OC, OP, DMP-1 were present in all experimental groups. Sclerostin was detected in OM-BMP-2 group only.

It was concluded that the investigated bioceramic scaffolds of hydroxylapatite and beta tricalcium phosphate support vitality and proliferation of human adipose-derived stem cells and induce ALP expression, therefore demonstrating promising osteogenic features for bone tissue engineering. On the other hand, an additional osteogenic stimulus (e.g. BMP-2) is required for the cells to accelerate the differentiation process.