Dentalis Nano Level Surface

The issue of bone-implant surface nanoroughness has received growing attention in the field of implant surface interaction in the last few years [1,2], as implant manufacturers – including dental implants – attempt to increase osseointerration via adjustments to surface topographies and implant chemistries [3]. Increased osteoconducivity and biocompatibility have been found to lead to better implant loading, as well as increased ossification expected even in areas with low bone mass and quality. [4,5]
In addition to the roughness of the implant surface, chemical modifications to the surface can also contribute to better bone creation and healing [2,6], as was previously shown in vivo and in vitro [7-11]. Presently, most implants are coated or saturated with crystals such as hydroxyapatite [12,13] or calcium phosphate [14-17].

Despite the importance of these chemical modifications, it is generally agreed upon that the texture of the implant surface is a significant factor in bone formation. [4,5] When compared to smooth-surface implants, creating surface roughness at the micrometer scale results in earlier fixation of the implants [4,5], seemingly due to improved protein absorption, cell adhesion, proliferation, and differentiation [1], though the role of roughness at the nanometer scale is still unclear. The interactions between hosts and “nano” implants are being actively studied and seem highly promising [12,13,15,16,18-20]; however, different chemical coating options make comparisons between different implants much harder, as they often contribute to the shape and roughness of the implant surface.

A recent study comparing an implant infused with small amounts of calcium (Ca) and phosphate (P) on a surface with nanoroughness qualities (CaP implant) to a control implant with smoother surface on the nano scale, processed through alumina-blasting/dual acid etching, and showed increased biomechanical fixation in the CaP implant. Another study compared these two implants on terms of bone cell activity [21].

Table I presents the differences in surface qualities as measured by a scanning electron microscope, and the most significant difference between the two implants appear to be the distance between the peaks on the surface – the CaP surface demonstrated much closer peaks than the control.
Quantification of the remaining blasting media on the implant surface is shown in Table II. Due to the sandblasting technique used in the control implant, residues of aluminum (Al) and silicon (Si) contaminated the implant surface.

Figure 1 shows SEM analysis of the surfaces of both implants. A different topography can be observed on the micrometric scale, with similar peak heights but shorter distances between the peaks in the CaP implant. The nanometric level shows significant differences between the samples: the surface of the control is almost flat, while the CaP surface demonstrates homogeneous nanoroughness. In addition, the calcium phosphate nanocrystals could not be observed, even in the highest magnification.

Fig. 1 Scanning electron microscope (SEM) analysis of the surfaces of control and CaP implants. Magnifications indicated on the left. Both implants enabled osteoblast adhesion and growth, without signs of cytotoxicity.

Fig. 2 shows the adhesion of osteoblasts to the surfaces of the implants. In both implants, the pattern is similar: after six hours, some cells are round, but some flatten and begin to present pseudopodes and vesicles, indicating metabolic activity and the beginning of adhesion. After 24 hours, most cells have flattened on the surface, and after 72 hours, all the cells are flat and mimic the surface topography, creating a veil of osteoblasts on the surface of the implants. Despite the similarities, the process accelerated on the CaP surface, and the osteoblasts showed more vesicles and pseudopods after only six hours, which is much less time than the control.

Fig 2. SEM analysis of Osteoblast adhesion to the surfaces of the implants. Magnifications and time indicated on the left.
Assessment of the proliferation and ALP activity of osteoblasts is presented in Fig.3. While the proliferation of osteoblasts on both surfaces was not significantly different, ALP-specific activity showed significantly higher values in the osteoblasts in contact with the CaP implant surface. The proliferation of osteoblast progenitors, human mesenchymal stem cells (hMSCs), was lower on the CaP surface, but the ALP-specific activity was once again higher in these cells.

Fig. 3 Proliferative activity assay (MTT assay) and alkaline phosphatase (ALP) specific activity of osteoblasts and human mesenchymal stem cells after 3,6,9 and 12 days in contact with surfaces of both implants. * values are significantly different (P < 0.01).

In conclusion, the CaP implants showed positive results correlated with cell surface nanoroughness compared to the control implant, and the low infusion resulted in nanocrystals that could not be detected with SEM even on high magnifications. The adhesion of osteoblasts to the surface of the CaP implant seem to appear earlier in “nano” implants than on the control implant, and despite the fact that no changes were observed in the number of osteoblasts on both surfaces and lower hMSCs on the CaP surface, ALP activity – a marker for osteoblasts’ activity – was higher on the CaP surface in both cell types.
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