Growth and Differentiation of Osteoblasts from Human Bone Fragments

ISO rules state that materials designed for biomedical applications must be submitted to bio-acceptability tests. During in vitro testing cultured cells should be as close as possible in origin and phenotype to the cells of interest. In studies on bone-biomaterial interfaces human osteoblasts are cells of choice. Objectives: This study describes cultivation procedures and methods for growth of osteoblasts from human bone fragments and a simple differentiation test to confirm the cell type. Methods: Human bone fragments were obtained during placement of implants. Fragments were immediately transferred into sterile test tubes containing 5ml DMEM with 2% PSF and transported to the cell culture laboratory. Fragments were placed into cell culture wells, covered with FBS and incubated at 37°C. After 2 hours DMEM containing 10% FBS and 1% PSF was added and the bony fragments were incubated at 37°C (humidified in 95% air + 5% CO₂). Cell wells were investigated daily by inverted microscopy to observe outgrowth of cells from the bony fragments. Once outgrowth was observed, media were replaced twice weekly and cells were grown to confluency. Confluent cells were passaged into two different cell wells and one well was used to confirm differentiation of osteoblasts. Human gingival fibroblasts were used as control. Both cell lines were cultivated in DMEM containing 50µg.ml^-1 ascorbic acid and 10mM glycerophosphate, grown to confluency and stained with Alizarin red (Sigma). The development of a red colour indicated presence of calcium mineral deposits and confirmed that cells differentiated into osteoblasts. Results: Provided bony fragments were free from bacterial and fungal contamination it was possible to cultivate osteoblasts from all samples gathered. The Alizarin red staining method proved to be a suitable test for confirmation of cell differentiation into osteoblasts. Conclusion: Cultivation and confirmation of differentiation of osteoblasts is possible by relative simple tissue culture procedures.