*3.3.2 Cell proliferation study*

Following the Cellular viability study viable cell count of 3T3-L1 cells on bare and coated substrates, the alloy was determined by staining them with 0.4% trypan blue at 1:5 ratios and counting them using a hemocytometer for cell proliferation study. Each experiment used a cell suspension with greater than 95% viability, as discussed in the experimental procedure. The difference in proliferation rates was found to be significant for alloy substrates and tissue culture plates (*p* < 0.05). Cell count on tissue culture plate (i.e. 110, 500, and 1500) was remarkably greater than that of alloy substrates (i.e. 48, 288, 365 for Ti6Al4V, 40, 218, 320 for CoCrMo and 32, 216, 310 for SS-316 L) after 2, 15 and 30 days respectively. A similar pattern with

#### *Tribological Behavior of Atmospheric Plasma Sprayed HA-CNT Coatings of Biomaterials DOI: http://dx.doi.org/10.5772/intechopen.103860*

a slight increase in cell count was also seen in reinforced HA-CNT coatings with different wt% ratios. The in vivo bone creation of CNT-coated Nanofillers was dramatically accelerated by greater bone mineral density and up-regulated osteogenic signs (ODN, OCN, BMP2) of bone-forming cells [46].

The reason for higher proliferation on HA-CNT coating as compared to the uncoated metallic substrate is justified by previous researchers that "hydroxyapatite once implanted into the body can easily react with physiological fluids and form a tenacious bond to hard and soft tissues through cellular activity". Another cause that gives an idea about the superior proliferation rate of coatings is the difference in surface roughness between porous HA-CNT coating and smooth bare metallic alloy substrates. Cytocompatibility and Osseointegration of the bone cement can be controlled by adjusting the MWCNT coating [47]. Furthermore, it may be observed from the results of this study that the proliferation rate on a tissue culture plate was comparatively higher than on all HA coatings due to their special design to enhance cell growth.
