Behavior of fetal rat osteoblasts cultured in vitro on the DP-bioactive glass substratum

It has been shown that new bone grew readily into the macropores of the DP-bioactive glass with chemical composition of Na2O 8.4%, CaO 40%, P2O5 and SiO2 39.6% in weight ratio. The bioactive glass would gradually dissolve in the in vivo environment, which would then be progressively replaced by the regenerated bone. Since the material was biodegradable, we wished to know whether the degradable product of DP-bioactive glass would be harmful to the osteoblast. This report described a dynamic analysis of early behavior of fetal rat osteoblasts cultured on a DP-bioactive glass substratum and expressed how the osteoblast attached onto the DP-bioactive glass substratum. Osteoblasts were isolated according to the method of Boonekamp with some modifications. Samples for morphological studies were seeded with a fell density of approximately 1 x 10(4) cells ml(-1), where 1 x 10(4) cells (1 ml) was added in each well and cultured for 8 h. Samples for cell population and rate of cell growth were seeded with a higher cell density of approximately 5 x 10(4) cells ml(-1), where 1 ml of cells was added in each well and cultured up to 6 days. During culturing, the six-well dishes were kept in an incubator with 95% air humidity and 5% CO2) at 37 degrees C. During the culturing period, half of the medium was replaced every 72 h. After culturing for a period of time, the substrata were carefully taken out from the culturing well dish. Both substrata and well dishes were fixed in 2.5% glutaraldehyde in 0.1 Fvl sodium cacodylate buffer for 2 h, rinsed with PBS (3 x 5 min) and dehydrated in a graded ethanol series. Optical microscopy and scanning electronic microscopy (SEM) were used for examination. Alkaline phosphotase stain was utilized for cell phenotype identification. Soda-lime glass and well dish ground (free-substratum) were included in the experiment as controlled groups. At the initial stage, the cell density of the free-substratum group was much lower than that of the DP-bioactive glass and soda-lime glass group. This was because there were more calcium ions on the free surface of DP-bioactive glass and soda-lime glass. In contrast, there were Ilo free calcium ions on the surface of the free-substratum group. This group had to absorb calcium ions from the cultural medium to its surface at the initial stage. We speculate that an incubation time is needed to take up calcium from the medium. which might be the reason for the lower cell population in the initial 12 h for the free-substratum group. The process of adhesion of osteoblasts in suspension to DP-bioactive glass substratum involved the following steps: ( ii calcium ion and serum proteins were absorbed onto the UP-bioactive glass substratum to form a protein template or fibronectin; (2) rounded cells then contacted the substratum and cell mitosis could also be observed elsewhere: (3) attachment of the osteoblasts to the substratum; and (4) spreading of the osteoblasts on the substratum, The fact that osteoblasts were stained with alkaline phosphate shows that the osteoblast did not transform into other types of cells. This observation indicated that the DP-bioactive glass, although constantly releasing calcium ions into the medium, would not inhibit the osteoblast growth and would not cause morphological transformation.