Isolation and Osteogenic Differentiation of Rat Periosteum-derived Cells

Selection of appropriate cultures having an osteogenic potential is a necessity if cell/biomaterial interactions are studied in long-term cultures. Osteoblastic cells derived from rat long bones or calvaria have the disadvantage of being in an advanced differentiation stage which results in terminal differentiation within 21 days. In this regard, less differentiated periosteum-derived osteoprogenitors could be more suitable. Periosteum-derived cells were isolated from the tibiae of adult Wistar rats (n = 12). The osteogenic potential with regard to alkaline phosphatase activity, morphology, nodule formation and mineralization was studied by culturing them in an osteogenic medium for up to 4 months. Seventy-five percent of the cultures (n = 9) did not show any increase in alkaline phosphatase activity nor nodule formation during long-term culture for up to 4 months. Nevertheless, in 25% of the cultures, alkaline phosphatase activity started from negligible (<5 mM pNP/mg protein) and increased towards approximately 50 mM pNP/mg protein. Three-dimensional nodule formation was observed at passages 3–5. In further passages (P5–P7), nodule formation capacity decreased and a diffuse mineralization pattern was observed. Suitable cultures with osteogenic capacity, can be selected at early passages based on the presence of cuboidal cells. These cells have the advantage of retaining their osteogenic potential even after prolonged cultivation (6–7 passages) before final differentiation occurs. Although periosteal cells are suitable for long term in vitro evaluation of biomaterials, the isolation and selection is time consuming. Hence, a more appropriate source to study cell/biomaterial interactions should be more convenient.