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the original seeding of the cells onto the device was performed manually, and in the subsequent experiments, the cells were seeded using a modified version of the device that enables automated seeding. the device was designed using a baseplate with 40 micrometre diameter circular wells, which were coated with fibronectin and collagen. a second plate with a silicon membrane was then bonded to the baseplate, allowing the cells to be seeded. the cells were cultured for up to 30 days in a medium consisting of rpmi 1640 supplemented with 10% fbs, 20% horse serum, 2mm l-glutamine, 100units/ml penicillin, 100μg/ml streptomycin, and 50μg/ml amphotericin b. the cells were maintained in a humidified incubator at 37°c with 5% co₂.

the cells were also stained with the anti-cd34 monoclonal antibody and sorted using a facscalibur flow cytometer. the cells were cultured in the absence of exogenous cytokines and were differentiated into the osteoblastic lineage using the calcium-phosphate method. the cells were cultured for a period of 21 days in the absence and presence of 10⁹ rat mesenchymal stem cells (rmsc) that were seeded in the wells of the microplates as an exogenous source of osteogenic soluble factors. both the cd34-positive and -negative cells were subjected to analysis using a bd facscalibur flow cytometer and cultured for up to 28 days in culture medium containing 10% fbs. the osteoblastic phenotype was confirmed by the expression of alkaline phosphatase and osteocalcin.

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