Only NIH/3T3 were able to colonize all structures within 14 days, which was likely caused by the high pressure of a high proliferation rate around the unstructured surfaces as well as a multi-layered growth or a higher secretion of ECM proteins, which eventually support the migration on an otherwise unsuitable surface. validity regarding the putative in vivo performance of implant material. < 0.05, ** < 0.01, *** < 0.001). By comparing both time points, the proliferation behavior of the individual cell populations on the different surfaces can be deduced. Proliferation was consistently the strongest for all those cell populations around the unstructured control surfaces, usually followed by growth on small spikes with the exception for MC3T3-E1, where large spikes had less negative effects (Physique 2C). The immortalized NIH/3T3 were significantly more proliferative than primary fibroblasts. Little growth, if not stagnation or reduction, in adherent cells on medium and large spikes was observed during investigation. Osteoblasts barely proliferated within 2 days around the investigated surfaces, and it seems not relevant whether cells are primary human or immortalized AZD4573 AZD4573 murine osteoblasts. The morphological changes of the adherent cells on spike structures compared to the AZD4573 unstructured control area could be determined by two parameters, the cell area and cell shape (aspect ratio). Regarding the cell area, it was found that there was a reduction in size around the spike structures, except for the NIH/3T3 on the small structures after 72 h (Physique 2D,E and Physique 3). The reduction in the cell area was particularly pronounced around the medium-sized and large structures. This turned out to be highly significant compared to the small structures after one day of attachment, except for primary osteoblasts. The cell shape of primary cells was dependent on spike size (Physique 2F,G). This dependence was more pronounced after 72 h than after 24 h. Both HGFib and NHOst were significantly longer around the medium spikes than on the small AZD4573 and large spikes (Physique 3). From the immortalized cells, only the NIH/3T3 were significantly longer on the small spikes than around the large spikes after 24 h but were less influenced by the presented spike distances than all other cell populations investigated concerning their morphological adaptation (Supplementary Physique S4). Open in a separate window Physique 3 Exemplary 3D reconstructions of HGFib, NIH/3T3, NHOst, and MC3T3-E1 after 72 h culture on flat control surfaces, small IB1 spikes, medium spikes, and large spikes. The cell reconstructions were based on the actin filament staining with phalloidin-TRITC and the surface topography was visualized using light reflection at 638 nm. A quantification and length determination of the FAs (focal adhesions) was only possible around the controls and small spikes, since hardly any FAs were detectable around the other structures. The FA length was divided into six categories (resolution limit 0.5 m) and the values of the small spikes were standardized to those of the controls. It became visible that for both time points and all cell types, the percentage of small FAs (0.5C1 m) on the small spikes was larger than around the control and the percentage of larger FAs (>1.5 m) decreased significantly (Determine 2H,I). The MC3T3-E1 had the highest percentage of small FAs with 334% after 24 h and 321% after 72 h. For the other cell types, this percentage was around 200% after 24 h and between 150% and 200% after 72 h. The percentage of FAs in the range of 1C1.5 m was similar to the control in all different cells. With increasing FA length, the percentage decreased to a similar extent in all cell types when compared to the control until it reached almost 0% (24 h) and 0C28% (72 h) for 3C3.5 m FAs. 2.3. The Spike Distance Influences Cell Migration of Peri-Implant Tissue AZD4573 Cells The representative images illustrate the different migration behavior of different cells on structured substrates over.