## 3 Results and Discussion  
In a previous study [28], nano-structured, micro-porous titanium surfaces were fabricated via hydrothermal treatment in a sodium hydroxide solution and evaluated for their blood-clotting properties and antibacterial properties against staphylococcus aureus and pseudomonas aeruginosa. The platelet adhesion and activation and whole blood-clotting characteristics were evaluated, and the adhesion, growth, and bacteria morphology were evaluated. Results indicated that NPTi surfaces demonstrated significantly decreased bacteria adhesion, proliferation, and biofilm formation (\(p\) < 0.05). NPTi surfaces displayed a reduction in growth of over 20% for Gram-positive bacteria and over 10% for Gram-negative bacteria. This reduction is a result of the surface topography of NPTi surfaces, which deforms the membrane of the bacteria cells and hence prevents their adhesion and subsequent growth. The blood-clotting characteristics (e.g., platelet adhesion and activation and whole blood clotting) were also evaluated for the surfaces. As compared to titanium, PTi and NPTi had higher rates of whole blood clotting after 15 min of clotting. However, after 45 min, all the surfaces had equivalent clotting. PTi and NPTi surfaces had significantly lower adhesion of platelets than Ti; however, the platelets had higher spreading for PTi and NPTi surfaces. Cell adhesion, proliferation, and differentiation are also influenced by surface properties like surface topography, surface chemistry, etc. Hence, in this study, cell adhesion, proliferation, and osteogenic differentiation of ADSCs were investigated. The surface topography of the surfaces was characterized using SEM (Figure 1) and is discussed elsewhere [28]. To explain in brief, Ti surfaces did not display any distinct features. The roughness could be due to the process used to clean the Ti surfaces. In the micro-porous surfaces, no topography change was observed at lower magnifications due to the HT process between unmodified (PTi) and modified (NPTi) surfaces. However, at higher magnifications, the effect of HT is visible in the form of nano-petal-like features and a \"web\"-like structure, which is formed because of the dissolution and precipitation mechanisms in the HT [25]. The HT parameters were determined to fabricate nanostructures while retaining the inherent micro-porous structure. XPS was performed to characterize the surface chemical composition (Table 1), which is explained in detail elsewhere [28]. In brief, titanium (Ti 2p3/2), oxygen (O1s), and carbon (C1s) were present on all the surfaces. Carbon presence was attributed to contamination from the XPS chamber and other surface impurities. NPTi surfaces had sodium (Na1s) present due to the HT in NaOH solution, which forms sodium titanate on the surfaces. Figure 1: SEM images of different surfaces at 100x and 5000x magnifications. For the long-term success of implants, it is crucial for the material to be conducive to cell growth and, hence, not be toxic towards them. A commercially available LDH assay was used to evaluate the cytotoxicity of different surfaces. LDH is an enzyme found in cells which, when released, is a sign of cell damage in the form of plasma membrane damage or lysis [11]. Higher LDH activity is an indicator of high cell damage, which shows the material is toxic. The manufacturer protocol was followed, and the absorbance was measured to calculate the LDH activity in the wells (Figure 2). Ti surfaces have been well established as a biocompatible material that is not cytotoxic. As expected, all the surfaces have similar LDH activity, demonstrating that they are not toxic to cells. NPTi surfaces are even a little less toxic than Ti surfaces, which indicates that HT is not only non-toxic towards cells but also removes some toxic matter from the surface. Similar behavior has been reported in studies involving HT treatment in an alkali medium [29].