Ring culture technique

The variable sizes of the implant disks obtained from two different manufacturers demanded an innovative culture system to ensure equal surface areas on both disks for the cell culture experiments. We achieved this by attaching constant diameter (5 mm) plastic cylinders to the disk surface. Disks were sterilized with absolute alcohol, and polystyrene cloning cylinders (Sigma) were attached onto the disks using vacuum grease. The enclosed surfaces on the disks were then coated with sterile 2% gelatin solution (Fig. 1).

Increased surface roughness in the 3M™ESPE™ MDIs

Scanning electron microscopy was used in a secondary electron mode under 10-kV acceleration voltage for producing the images to observe the surface topography, and it showed an increased surface roughness in the 3M™ESPE™ MDIs as compared with Ankylos® (Fig. 2).

Increased proliferation of C2C12 cells grown on 3M™ESPE™ MDI disks

We first examined the proliferation of C2C12 cells treated with BMP-2, a pro-osteogenic cytokine, or without BMP-2 treatment on both types of disks. Ten thousand C2C12 cells were plated, and on the following day, the medium was supplemented with 300 ng/ml of BMP-2. Cells were grown for 3 days, stained with the nuclear stain H33258, and imaged using fluorescence microscopy. Counting of cell nuclei revealed an increased proliferation of the C2C12 cells grown on 3M™ESPE™ MDI disks under both conditions, when treated with BMP-2 or without any treatment (Fig. 3).

Disk type does not affect osteogenic differentiation

C2C12 myoblastic cells were transfected with BMP-2. These cells express high levels of ALPL when compared with the control (untransfected) group (Fig. 4a). ALPL zymography showed a more intense band indicating very high expression of functional ALPL protein in the stably transfected cells (Fig. 4b). The transfected cells were then seeded onto each type of disks (15,000 cells/disk) and were cultured for 3 days. Immunostaining using a goat anti-mouse ALPL antibody revealed a significantly higher number of ALPL-positive cells on the 3M™ESPE™ MDI disks in comparison to those on the Ankylos® disks. Interestingly, when the number of ALPL-positive cells was normalized to the total cell number, no differences were observed. This finding suggests that the increase of ALPL-positive cells was not due to an increased cell differentiation, but because of an increased cell proliferation (Fig. 4c).

Increased proliferation of MC3T3-E1 cells and extracellular matrix mineralization on 3M™ESPE™ MDI disks

Pre-osteoblastic MC3T3-E1 cells were plated on each implant disk (40,000 cells/disk) and were differentiated with mineralization medium for 12 days. Quantification of cells after nuclear staining by H33258 revealed an increased number of cells on the 3M™ESPE™ MDI disks (Fig. 5a). Measurement of cell viability by the reduction of Alamar blue® after 3 days of culture of MC3T3-E1 cells further supported an increase of cell proliferation on the 3M™ESPE™ MDI disks (Fig. 5b).

In order to assess the ability of the system to promote ECM mineralization, MC3T3-E1 cells were plated at equal densities on each disk type and were grown in the presence of differentiation medium for 12 days. Calcein (binds to calcium salts) staining demonstrated an increased mineral deposition on the surface of the 3M™ESPE™ MDI disks when compared with that on the Ankylos® disks. Increased cell proliferation in the 3M™ESPE™ MDI disks cultures may explain the increase in ECM mineralization (Fig. 5c).